Cancer stem cells (CSCs), which drive tumor growth, are known to be resistant to standard chemotherapy and radiation treatment. This raises a significant unmet need to find therapies that can target CSCs within tumors because these cells are responsible for recurrence, the primary cause of patient mortality. However, one of the technical challenges of working with CSCs is that they are not stable outside the tumor environment and are not easy to grow in culture media. Hence, stable sibling cell lines that were induced into epithelial-to-mesenchymal transdifferentiation (EMT) to stably propagate CSC-enriched populations were used to screen a library of 300,718 compounds from the Molecular Libraries Small Molecule Repository (MLSMR). Several classes of selective inhibitors of CSCs were identified. The use of isogenic control cell lines for the secondary validation assays minimized the probability of false hits advancing along the critical path to probe development. Of these, 19 compounds were chosen based on their selectivity, potency, and chemical tractability and were retested in the primary screen and secondary assays. Three scaffolds were prioritized to develop potential probes. One of these compounds (ML243) displayed greater than 32-fold selective inhibition of the breast CSC-like cell line (HMLE_shECad) over the control cell line (HMLE_shGFP). The probe (ML243) was screened against a panel of 68 targets that are commonly used in drug discovery for lead profiling and found active in only one assay.