BACKGROUND: Disease recurrence and progression of ovarian cancer is a common event, which is accompanied by the development of platinum-resistant or refractory disease. The presence of chemo-resistant Cancer Stem Cells (CSCs) contribute to tumor propagation, maintenance, and treatment resistance of this difficult to treat disease. We have developed ChemoID, a cytotoxic synergy assay against CSCs that identifies the most effective chemotherapy treatment from a panel of FDA-approved chemotherapies using fresh cancer biopsies. PATIENTS AND METHODS: Ascites or interventional radiology biopsies were collected under physician order from 78 consecutive patients affected by 3rd relapsed ovarian cancer. Test results from the assay were used when possible to treat patients with the highest cell kill drugs, taking into consideration their health status and using dose reductions, if needed. A chart analysis and review of CT and PET scans were performed to determine patients' outcomes for tumor response, Progression-Free Survival (PFS), and Overall Survival (OS). RESULTS: We observed that recurrent ovarian cancer patients treated with high-cell kill chemotherapy agents guided by the CSCs drug response assay had an improvement in their median PFS and OS when compared to historical median PFS and OS and/or when compared to patients who could not receive high cell kill chemotherapies (PFS low cell kill 3.5 months vs. high cell kill 12.0 months; OS low cell kill 6.0 months vs. high cell kill 15.0 months). CONCLUSION: This data indicates that the drug cytotoxicity assay aimed at targeting CSCs may be a useful tool for optimizing treatment selection when first-line therapy fails, and when there are multiple clinically-acceptable and -equivalent treatments available.