One major aspect of personalised medicine is patient-to-patient variation in drug toxicity and response. Pharmacogenomics attempts to address this issue by identifying genetic factors that contribute to this variation. The Collaborative Cross (CC) mice consist of hundreds of octo-parental recombinant inbred lines, which account for greater than 90% of known variations in mice. This genetic reference population resembles the diversity seen in humans. Here we have established a cell culture collection of immortalised primary fibroblasts from the CC mice to overcome the problems of animal husbandry and to harness the high throughput power of cell culture models. Using the C4 collection, we have treated multiple cell lines with five different cytoskeleton-active drugs. Treated fibroblasts were stained for F-actin with phalloidin and DAPI and imaged using high throughput microscopy which were further analysed with a custom designed analysis pipeline. Different strains show varied phenotypic response to the five cytoskeleton-active drugs. These phenotype data can then be applied for quantitative trait locus mapping to find genes of interest that modulate the drug response.