INTRODUCTION: Endometriosis is an estrogen-dependent, chronic, pro-inflammatory disease that develops in 6-10% of women of reproductive age. Symptoms include chronic pelvic pain, dysmenorrhea and subfertility. Current therapeutic interventions include hormone modifiers, non-steroidal anti-inflammatories and surgery. Unfortunately, these interventions are often short term and non-curative highlighting a significant need for the discovery of new compounds that disrupt the estrogen-driven pathways in endometriosis. To achieve this, a high-throughput screen (HTS) of 3,500 clinically approved drugs was performed using immortalized human endometriosis stromal cell lines. METHODS: Stromal cells were seeded into 384 well plates and screened with a single dose of library drug [10 µM] in the presence or absence of estradiol-17β. Three days later, the cells were fixed, stained and imaged using the Cell-Insight CX7 High Content Screen platform where a nuclear count was performed using HCS Studio Cell Analysis Software. RESULTS: Drug hits were identified as those that caused a >70% inhibition during estrogen treatment compared to vehicle. This process identified 40 unique drug hits for each cell line. These drug hits included a diverse range of compound types including anti-cancer, anti-diabetic, anti-histamine and antibiotics. CONCLUSION: The HTS on endometriosis stromal cells identified 40 drugs that interfered with estrogen driven cell growth and targeted novel pathways down-stream of the estrogen receptors. These findings highlight novel mechanisms that may contribute to endometriosis lesion formation and demonstrate strong heterogeneity of cell signalling between cell lines. This study also demonstrates the value of HTS for endometriosis therapeutic discovery and the potential for repurposing clinically approved drugs for personalised endometriosis therapy.