Idiopathic Pulmonary fibrosis (IPF) is a devastating disease with no treatment that can stop or reverse fibrosis. At AstraZeneca we are committed to developing our understanding of the aberrant processes driving fibrosis. Myofibroblasts play a central role in this process, with transforming growth factor ?1 (TGF-?1) inducing fibroblast-to-myofibroblast transition (FMT) that results in an excessive expression of ?-smooth muscle actin (?SMA), a robust marker of myofibroblast differentiation, and deposition of extracellular matrix. Setting up a high throughput assay for screening potential drug candidates that can impact on FMT has proved difficult because the required level of automation may introduce mechanical stress which can lead to cell detachment and increased variability, particularly when using primary human lung fibroblasts to develop an assay as relevant as possible. To attempt this, multiple parameters required optimization. Shortly, cells were plated and FMT was induced through TGF-?1 stimulation. Multiple automated liquid handling equipment, 384-well plate variants, cell numbers, TGF-?1 concentration and washing protocol were optimised through analysis of ?SMA protein expression using fluorescence-based imaging. Minimum discrepancy ratio (MDR) statistics was used to assess assay performance. The assay was validated by running 12 compounds on three separate days. The fully optimised assay performed with a MDR=2.6 using two technical replicates on one occasion. A MDR<4 is considered to reflect a well behaving assay with reproducible potency estimates. In conclusion a stable high throughput FMT assay in primary human lung fibroblasts has been optimised to be run in 384 well format.