diff --git a/modules/nf-core/ashlar/main.nf b/modules/nf-core/ashlar/main.nf
new file mode 100644
index 00000000000..2316b024391
--- /dev/null
+++ b/modules/nf-core/ashlar/main.nf
@@ -0,0 +1,52 @@
+process ASHLAR {
+    tag '$meta.id'
+    label 'process_single'
+
+    conda "bioconda::ashlar=1.17.0"
+    container "${ workflow.containerEngine == 'singularity' && !task.ext.singularity_pull_docker_container ?
+        'https://depot.galaxyproject.org/singularity/ashlar:1.17.0--pyh5e36f6f_0' :
+        'biocontainers/ashlar:1.17.0--pyh5e36f6f_0' }"
+
+    input:
+    tuple val(meta), path(images)
+    path(opt_dfp)
+    path(opt_ffp)
+
+    output:
+    tuple val(meta), path("*.ome.tif"), emit: tif
+    path "versions.yml"               , emit: versions
+
+    when:
+    task.ext.when == null || task.ext.when
+
+    script:
+    def args          = task.ext.args           ?: ''
+    def prefix        = task.ext.prefix         ?: "${meta.id}"
+    def dfp           = opt_dfp                 ? "--dfp $opt_dfp" : ""
+    def ffp           = opt_ffp                 ? "--ffp $opt_ffp" : ""
+    def num_files     = images instanceof List  ? images.size()    : 1
+    def opt_dfp_size  = opt_dfp instanceof List ? opt_dfp.size()   : 1
+    def opt_ffp_size  = opt_ffp instanceof List ? opt_ffp.size()   : 1
+    def dfp_validated = (opt_dfp_size == 0 || opt_dfp_size == 1 || opt_dfp_size == num_files) ? true : false
+    def ffp_validated = (opt_ffp_size == 0 || opt_ffp_size == 1 || opt_ffp_size == num_files) ? true : false
+
+    if ( !dfp_validated ) { error "Please input only zero, one, or N dfp files, where N is the number of input images" }
+    if ( !ffp_validated ) { error "Please input only zero, one, or N ffp files, where N is the number of input images" }
+
+    """
+
+    ashlar \\
+        -o ${prefix}.ome.tif \\
+        $images \\
+        $args \\
+        $dfp \\
+        $ffp
+
+    sed -i -E 's/UUID="urn:uuid:[[:xdigit:]]{8}-[[:xdigit:]]{4}-[[:xdigit:]]{4}-[[:xdigit:]]{4}-[[:xdigit:]]{12}"/                                                    /g' ${prefix}.ome.tif
+
+    cat <<-END_VERSIONS > versions.yml
+    "${task.process}":
+        ashlar: \$(ashlar --version 2>&1 | sed 's/^.*ashlar //; s/Using.*\$//' )
+    END_VERSIONS
+    """
+}
diff --git a/modules/nf-core/ashlar/meta.yml b/modules/nf-core/ashlar/meta.yml
new file mode 100644
index 00000000000..244e9726616
--- /dev/null
+++ b/modules/nf-core/ashlar/meta.yml
@@ -0,0 +1,42 @@
+name: "ashlar"
+description: Alignment by Simultaneous Harmonization of Layer/Adjacency Registration
+keywords:
+  - image_processing
+  - alignment
+  - registration
+tools:
+  - "ashlar":
+      description: "Alignment by Simultaneous Harmonization of Layer/Adjacency Registration"
+      homepage: "https://labsyspharm.github.io/ashlar/"
+      documentation: "https://labsyspharm.github.io/ashlar/"
+      doi: "10.1093/bioinformatics/btac544"
+      licence: ["MIT"]
+
+input:
+  - meta:
+      type: map
+      description: |
+        Groovy Map containing sample information
+        e.g. [ id:'test' ]
+  - images:
+      type: file
+      description: Overlapping tile image data in formats from most commercial microscopes
+      pattern: "*.{ome.tiff,ome.tif,rcpnl,btf,nd2,tiff,tif,czi}"
+output:
+  - meta:
+      type: map
+      description: |
+        Groovy Map containing sample information
+        e.g. [ id:'test' ]
+  - tif:
+      type: file
+      description: A pyramidal, tiled OME-TIFF file created from input images.
+      pattern: "*.ome.tif"
+  - versions:
+      type: file
+      description: File containing software versions
+      pattern: "versions.yml"
+
+authors:
+  - "@RobJY"
+  - "@jmuhlich"
diff --git a/tests/config/pytest_modules.yml b/tests/config/pytest_modules.yml
index 78874595b36..7a4308fb1d4 100644
--- a/tests/config/pytest_modules.yml
+++ b/tests/config/pytest_modules.yml
@@ -134,6 +134,10 @@ ascat:
   - modules/nf-core/ascat/**
   - tests/modules/nf-core/ascat/**
 
+ashlar:
+  - modules/nf-core/ashlar/**
+  - tests/modules/nf-core/ashlar/**
+
 assemblyscan:
   - modules/nf-core/assemblyscan/**
   - tests/modules/nf-core/assemblyscan/**
diff --git a/tests/config/test_data.config b/tests/config/test_data.config
index 5887ad5fe46..fa5d826cb12 100644
--- a/tests/config/test_data.config
+++ b/tests/config/test_data.config
@@ -671,6 +671,8 @@ params {
                 cycif_tonsil_cycle1     = "${params.test_data_base}/data/imaging/ome-tiff/cycif-tonsil-cycle1.ome.tif"
                 cycif_tonsil_cycle2     = "${params.test_data_base}/data/imaging/ome-tiff/cycif-tonsil-cycle2.ome.tif"
                 cycif_tonsil_cycle3     = "${params.test_data_base}/data/imaging/ome-tiff/cycif-tonsil-cycle3.ome.tif"
+                cycif_tonsil_dfp        = "${params.test_data_base}/data/imaging/ome-tiff/cycif-tonsil-dfp.ome.tif"
+                cycif_tonsil_ffp        = "${params.test_data_base}/data/imaging/ome-tiff/cycif-tonsil-ffp.ome.tif"
             }
             'registration' {
                 markers                 = "${params.test_data_base}/data/imaging/registration/markers.csv"
diff --git a/tests/modules/nf-core/ashlar/main.nf b/tests/modules/nf-core/ashlar/main.nf
new file mode 100644
index 00000000000..278bc3195c7
--- /dev/null
+++ b/tests/modules/nf-core/ashlar/main.nf
@@ -0,0 +1,90 @@
+#!/usr/bin/env nextflow
+
+nextflow.enable.dsl = 2
+
+include { ASHLAR } from '../../../../modules/nf-core/ashlar/main.nf'
+include { ASHLAR as ASHLAR_TILE } from '../../../../modules/nf-core/ashlar/main.nf'
+
+workflow test_ashlar_1_file {
+
+    input_list =  [ [ id:'test_all' ],
+               [file(params.test_data['imaging']
+                                     ['ome-tiff']
+                                     ['cycif_tonsil_cycle1'], checkIfExists: true)] ]
+
+    ASHLAR ( input_list, [], [] )
+
+}
+
+workflow test_ashlar_all_files {
+
+    input_list =  [ [ id:'test_all' ],
+               [file(params.test_data['imaging']
+                                     ['ome-tiff']
+                                     ['cycif_tonsil_cycle1'], checkIfExists: true),
+                file(params.test_data['imaging']
+                                     ['ome-tiff']
+                                     ['cycif_tonsil_cycle2'], checkIfExists: true),
+                file(params.test_data['imaging']
+                                     ['ome-tiff']
+                                     ['cycif_tonsil_cycle3'], checkIfExists: true)] ]
+
+    ASHLAR ( input_list, [], [] )
+
+}
+
+workflow test_ashlar_all_files_tile_size {
+
+    input_list =  [ [ id:'test_all' ],
+               [file(params.test_data['imaging']
+                                     ['ome-tiff']
+                                     ['cycif_tonsil_cycle1'], checkIfExists: true),
+                file(params.test_data['imaging']
+                                     ['ome-tiff']
+                                     ['cycif_tonsil_cycle2'], checkIfExists: true),
+                file(params.test_data['imaging']
+                                     ['ome-tiff']
+                                     ['cycif_tonsil_cycle3'], checkIfExists: true)] ]
+
+    ASHLAR_TILE ( input_list, [], [] )
+
+}
+
+workflow test_ashlar_all_files_dfp_ffp {
+
+    input_list =  [ [ id:'test_all' ],
+               [file(params.test_data['imaging']
+                                     ['ome-tiff']
+                                     ['cycif_tonsil_cycle1'], checkIfExists: true),
+                file(params.test_data['imaging']
+                                     ['ome-tiff']
+                                     ['cycif_tonsil_cycle2'], checkIfExists: true),
+                file(params.test_data['imaging']
+                                     ['ome-tiff']
+                                     ['cycif_tonsil_cycle3'], checkIfExists: true)] ]
+
+    ASHLAR ( input_list, [file(params.test_data['imaging']
+                                               ['ome-tiff']
+                                               ['cycif_tonsil_dfp'], checkIfExists: true)],
+                         [file(params.test_data['imaging']
+                                               ['ome-tiff']
+                                               ['cycif_tonsil_ffp'], checkIfExists: true)] )
+
+}
+
+workflow test_ashlar_1_file_dfp_ffp {
+
+    input_list =  [ [ id:'test_all' ],
+               [file(params.test_data['imaging']
+                                     ['ome-tiff']
+                                     ['cycif_tonsil_cycle1'], checkIfExists: true)] ]
+
+    ASHLAR ( input_list, [file(params.test_data['imaging']
+                                               ['ome-tiff']
+                                               ['cycif_tonsil_dfp'], checkIfExists: true)],
+                         [file(params.test_data['imaging']
+                                               ['ome-tiff']
+                                               ['cycif_tonsil_ffp'], checkIfExists: true)] )
+
+}
+
diff --git a/tests/modules/nf-core/ashlar/nextflow.config b/tests/modules/nf-core/ashlar/nextflow.config
new file mode 100644
index 00000000000..9b566dd3953
--- /dev/null
+++ b/tests/modules/nf-core/ashlar/nextflow.config
@@ -0,0 +1,9 @@
+process {
+
+    publishDir = { "${params.outdir}/${task.process.tokenize(':')[-1].tokenize('_')[0].toLowerCase()}" }
+
+    withName: ASHLAR_TILE {
+        ext.args = "--tile-size 512"
+    }
+
+}
diff --git a/tests/modules/nf-core/ashlar/test.yml b/tests/modules/nf-core/ashlar/test.yml
new file mode 100644
index 00000000000..be9450cc22d
--- /dev/null
+++ b/tests/modules/nf-core/ashlar/test.yml
@@ -0,0 +1,44 @@
+- name: ashlar test_ashlar_1_file
+  command: nextflow run ./tests/modules/nf-core/ashlar -entry test_ashlar_1_file -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/ashlar/nextflow.config
+  tags:
+    - ashlar
+  files:
+    - path: output/ashlar/test_all.ome.tif
+      md5sum: ae98ac74e3c818bb26c99ba4b9c2dd51
+    - path: output/ashlar/versions.yml
+
+- name: ashlar test_ashlar_all_files
+  command: nextflow run ./tests/modules/nf-core/ashlar -entry test_ashlar_all_files -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/ashlar/nextflow.config
+  tags:
+    - ashlar
+  files:
+    - path: output/ashlar/test_all.ome.tif
+      md5sum: 5f4165203545b7efe04ed5f4a6d5ac9d
+    - path: output/ashlar/versions.yml
+
+- name: ashlar test_ashlar_all_files_tile_size
+  command: nextflow run ./tests/modules/nf-core/ashlar -entry test_ashlar_all_files_tile_size -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/ashlar/nextflow.config
+  tags:
+    - ashlar
+  files:
+    - path: output/ashlar/test_all.ome.tif
+      md5sum: acee3aeaf7ea088062b2c15eff72dfb4
+    - path: output/ashlar/versions.yml
+
+- name: ashlar test_ashlar_all_files_dfp_ffp
+  command: nextflow run ./tests/modules/nf-core/ashlar -entry test_ashlar_all_files_dfp_ffp -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/ashlar/nextflow.config
+  tags:
+    - ashlar
+  files:
+    - path: output/ashlar/test_all.ome.tif
+      md5sum: 5f4165203545b7efe04ed5f4a6d5ac9d
+    - path: output/ashlar/versions.yml
+
+- name: ashlar test_ashlar_1_file_dfp_ffp
+  command: nextflow run ./tests/modules/nf-core/ashlar -entry test_ashlar_1_file_dfp_ffp -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/ashlar/nextflow.config
+  tags:
+    - ashlar
+  files:
+    - path: output/ashlar/test_all.ome.tif
+      md5sum: ae98ac74e3c818bb26c99ba4b9c2dd51
+    - path: output/ashlar/versions.yml