Abstract The KRAS fraction in the plasma membrane (PM) correlates with activation of the MAPK pathway and subsequent cellular proliferation. Understanding KRAS interaction with the PM constitutes a challenge given the complexity of the cellular environment. To gain insight on key components necessary for KRAS signal transduction at the membrane, we make use of reconstituted liposomes and giant unilamellar vesicles as synthetic membranes. Using surface plasmon resonance (SPR) spectroscopy, we demonstrate that KRAS and RAF1 domains interact with these membranes primarily through electrostatic interactions with negatively charged lipids reinforced by additional interactions involving phosphatidyl ethanolamine and cholesterol. RAF1 52-188 (RBDCRD) interacts with the membrane significantly more strongly than the isolated RBD or CRD domains and synergizes KRAS membrane partitioning. Calmodulin and PDE6 delta, but not galectin3, passively sequester KRAS and prevent it from binding to the PM. RAF1 RBDCRD interacts with membranes preferentially at non-raft lipids domains. The carboxyterminal O-methylation is crucial for KRAS membrane localization. These results contribute to a better understanding of how KRAS-membrane interaction can be tuned by multiple factors. Citation Format: Bindu Lakshman, Simon Messing, Eva M. Schmid, Jeffrey D. Clogston, William K. Gillette, Dominic Esposito, Bailey Kessing, Daniel A. Fletcher, Dwight V. Nissley, Frank McCormick, Andrew G. Stephen, Frantz L. Jean-Francois. Quantitative biophysical analysis defining key components modulating KRAS recruitment to the plasma membrane [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A10.