The PTEN tumor suppressor gene is one of the most commonly mutated genes in human cancer. Because inactivation of PTEN is a somatic event, PTEN mutations represent an important genetic difference between cancer cells and normal cells and therefore a potential anticancer drug target. However, it remains a substantial challenge to identify compounds that target loss-of-function events such as mutations of tumor suppressors. In an effort to identify small molecules that preferentially kill cells with mutations of PTEN, the authors developed and implemented a high-throughput, paired cell-based screen composed of parental HCT116 cells and their PTEN gene-targeted derivatives. From 138 758 compounds tested, two hits were identified, and one, N'-[(1-benzyl-1H-indol-3-yl)methylene]benzenesulfonohydrazide (CID1340132), was further studied using a variety of cell-based models, including HCT116, MCF10A, and HEC1A cells with targeted deletion of either their PTEN or PIK3CA genes. Preferential killing of PTEN and PIK3CA mutant cells was accompanied by DNA damage, inhibition of DNA synthesis, and apoptosis. Taken together, these data validate a cell-based screening approach for identifying lead compounds that target cells with specific tumor suppressor gene mutations and describe a novel compound with preferential killing activity toward PTEN and PIK3CA mutant cells.