ARTICLE ABSTRACTMost of the currently used cancer chemotherapies are based on compounds that inhibit general cellular mechanisms, such as DNA replication or tubulin function, and lack specificity in relation to features of the cancer cell. Recent advances in genomic studies have increased our knowledge of tumor cell biology, and a panoply of new targets have been postulated. This has provided an opportunity to develop and validate drugs that specifically target cancer cells through their unique genetic characteristics. Identification of MUC16/CA125 both as a marker and a driver of transformation led us to design a target-based high-content screen to identify and classify compounds that exhibit differential effect on MUC16-expressing cells. We developed a coculture assay in 384-well plate containing isogenic ovarian cancer cells that are positive or negative for the MUC16 protein. High-throughput screening of our small molecule pilot library led to the identification of compounds preferentially cytotoxic to MUC16+ or MUC16− cells, using a Preferential Score analysis. We compared screening results in both A2780 and SK-OV-3 ovarian cancer cells in single and coculture settings. We also identified compounds that were cytotoxic for both types of ovarian cancer cells regardless of the MUC16 status. Compounds that were preferentially targeting MUC16 cells were subsequently confirmed by caspase-induction assays. The isogenic, dual-color fluorescence strategy is an innovative approach that can effectively identify novel drug candidates, selectively targeting cancer cells that have unique molecular properties. Mol Cancer Ther; 10(10); 1939–48. ©2011 AACR.