Abstract Replication Protein A (RPA) is a major regulator of checkpoint activation and enhanced DNA repair in cancer cells. In response to genotoxic stress, the RPA complex binds to and protects ssDNA while serving as a scaffold to recruit critical checkpoint and DNA-damage response proteins through the N-terminal region of the 70 kDa subunit of RPA (RPA70N). RNAi against RPA has shown an expected toxicity against cancer cell lines. However, specific disruption of the RPA protein-protein interactions mediated by the RPA70N domain has the potential to produce selective killing of cancer cells without of cytotoxicity due to interference with its ssDNA-binding function. In order to accurately examine the therapeutic relevance of the inhibition of RPA function, we have sought to discover potent probe molecules that disrupt the interactions between RPA70N and its binding partners. Here we describe the discovery of molecules to probe RPA function using complementary fragment-based and traditional high-throughput screening techniques. SAR studies and structure-based design concepts used to optimize the lead series of interest will be discussed along with the biochemical and cellular results obtained with the compounds. Citation Format: Alex G. Waterson, Phillip Kennedy, James D. Patrone, Nicholas F. Pelz, Andreas O. Frank, Bhavatarini Vangamudi, DeMarco V. Camper, Elaine M. Souza-Fagundes, Michael D. Feldkamp, Edward T. Olejniczak, Olivia W. Rossanese, Walter J. Chazin, Stephen W. Fesik. Discovery of probes to evaluate the disruption of the protein-protein interactions mediated by RPA70N. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3695. doi:10.1158/1538-7445.AM2015-3695