Abstract KRAS4b is a major driver of cancers of the pancreas, lung, and colon, and until recently was considered undruggable. However, recent discoveries have revealed that molecules can target KRAS directly, and that our understanding of KRAS is incomplete. To exploit this new appreciation of KRAS druggability and biology, we have developed several protein-protein interaction (PPI) assays to target KRAS activity in live cells. Here we report on a campaign where we screened a 20,000-compound library against full length KRAS4b-G12D and RAF1 interactions in live HEK293 cells using the NanoBRET platform. From the list of screening hits, we identified several structurally related active compounds with dose-dependent, albeit weak, inhibition of the PPI signal. We expanded these active compounds to a more complete list of structural analogs which we tested in a full dose response. From this expanded group of compounds, we identified several with low µM IC50s. Follow up with these compounds in secondary assays revealed promising biological readouts, including inhibition of downstream pERK levels in G12D mutant PANC1 cells. In summary, by using well-designed live cell assays, we have identified promising leads, which we are pursuing with mechanism of action studies. Citation Format: John Columbus, Thomas J. Turbyville, Vanessa Wall, Dominic Esposito, David A. Barda, Sheng-Bin Peng. A live-cell, protein-protein interaction assay identified inhibitors of KRAS4b-G12D interaction with full-length RAF1 abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-062.