Synthetic lethality is a successful strategy employed to develop selective chemotherapeutics against cancer cells. Inactivation of RAD52 is synthetically lethal to homologous recombination (HR) deficient cancer cell lines. Replication protein A (RPA) recruits RAD52 to repair sites, and the formation of this protein-protein complex is critical for RAD52 activity. To discover small molecules that inhibit the RPA:RAD52 protein-protein interaction (PPI), we screened chemical libraries with our newly developed Fluorescence-based protein-protein Interaction Assay (FluorIA). Eleven compounds were identified, including FDA-approved drugs (quinacrine, mitoxantrone, and doxorubicin). The FluorIA was used to rank the compounds by their ability to inhibit the RPA:RAD52 PPI and showed mitoxantrone and doxorubicin to be the most effective. Initial studies using the three FDA-approved drugs showed selective killing of BRCA1-mutated breast cancer cells (HCC1937), BRCA2-mutated ovarian cancer cells (PE01), and BRCA1-mutated ovarian cancer cells (UWB1.289). It was noteworthy that selective killing was seen in cells known to be resistant to PARP inhibitors (HCC1937 and UWB1 SYr13). A cell-based double-strand break (DSB) repair assay indicated that mitoxantrone significantly suppressed RAD52-dependent single-strand annealing (SSA) and mitoxantrone treatment disrupted the RPA:RAD52 PPI in cells. Furthermore, mitoxantrone reduced radiation-induced foci-formation of RAD52 with no significant activity against RAD51 foci formation. The results indicate that the RPA:RAD52 PPI could be a therapeutic target for HR-deficient cancers. These data also suggest that RAD52 is one of the targets of mitoxantrone and related compounds.