This probe report describes the identification and development of an inhibitor of Bcl-B, a member of the Bcl-2 family. The Bcl-2 family plays a prominent role in apoptosis. Bcl-2 is over-expressed in some cancers, allowing cells to continue proliferating. Thus, small molecule inhibitors of the anti-apoptotic Bcl-2 family members with their apoptotic partners would be useful for enhancing cancer chemotherapy. We developed a multiplexed bead-based flow cytometry high-throughput assay based on the disruption of the binding of a fluorescently labeled-BH3 peptide of Bim to the six anti-apoptotic Bcl-2 family members: Bcl-XL, Bcl-W, Bcl-B, Bfl-1, and Mcl-1 and Bcl-2 (the eponymous founding member of the Bcl-2 family). Using this assay, we screened each of 200,000 compounds in the NIH Molecular Libraries Small Molecule Repository (MLSMR) simultaneously (in multiplexed format) against all six Bcl-2 family members for potential regulators of these crucial peptide-protein interactions. We were able to develop a potent (368 nM IC50) inhibitor of the interaction of the Bim peptide with Bcl-B that was at least 127-fold selective over the Bim-Bcl-XL interaction. It was also shown to be selective against the other family members (Bcl-W, Bcl-2, Bfl-1, Mcl-1).