Ras proteins function as binary switches in order to mediate a wide range of signaling events and are one of the most frequently mutated oncoproteins in human cancers. However, progress in targeting Ras has been slow owing to the lack of obvious druggable sites. Recently, numerous binding proteins have been developed that probe Ras function, which have identified some allosteric sites and have opened new avenues for drug discovery. Affimers are now an established class of binding proteins. They consist of two variable regions that confer molecular recognition with target proteins and molecules. We have isolated Affimers against KRas using phage display. From the phage screen, seven unique binders were isolated and tested in SOScat catalyzed nucleotide exchange reaction to identify inhibitors of nucleotide exchange. Three Affimers, K3, K6 and K37, were the most potent inhibitors of this reaction, with K3 also inhibiting Ras interaction with the Ras binding domain of Raf. To understand the molecular mechanisms of inhibiting both nucleotide exchange and interaction with Raf, the X-ray crystal structure of Affimer K3-KRas complex was solved to 2.1 Å resolution. The Affimer interacts with Switch II region of KRas, causing the α-2 helix region to shift outwards to reveal a hydrophobic pocket that has not been targeted by any other non-antibody binding proteins. It is speculated that the shift in the switch II domain will cause a steric clash with the Ras binding domain of Raf. These results show that Affimers can select for protein conformations not previously identified by other reagents and may aid in further developing Ras binding compounds. Citation Format: Ajinkya Rao, Kataryzna Haza, Chi Trinh, Thomas Edwards, Darren Tomlinson. Inhibition of Ras using Affimers [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 B33.