The binding site of indole hit 62 was explored by LC-MS/MS analysis of crosslinked KRas4B G12D . Three modified peptides were identified corresponding to crosslinking on three different residues on the same peptide (57DTAGQE*E*Y*SAM ox R68, Figure ). These residues, E62, E63 and Y64, lie at the loop of the switch I/II region, directly adjacent to the binding site of ligands reported previously (e.g. 64, PDB ID:4EPY, Figure ). This pocket is of functional significance in interacting with SOS1/2, the guanine nucleotide exchange factors (GEFs), which promote formation of the active KRas state. Thus, the PhABit platform enabled expedient identification of fragment binders to a protein of low tractability, and provided insight into the binding site of hit PhABits without the need for recourse to NMR and crystallography. A challenge associated with KRas has been access to assays to enable efficient screening of compounds libraries. We explored the use of the photoaffinity displacement assay to measure IC50 values of compounds of interest. A range of concentrations of previously reported KRas binders 64 and 65 (Figure ) were incubated with KRas4B G12D (1 µM) and hit PhABit 62 (1 mM) before irradiation. The crosslinking yields were determined by LC-MS to afford the expected concentration-dependent decrease in crosslinking (Figure ). The generated IC50 values of 170 µM (64) and 90 µM (65) were consistent with reported values (64 = 340 µM, and 65 = 190 µM), which were determined by HSQC NMR using N-labelled KRas G12D . This displacement assay therefore provides an efficient method for screening ligands against the switch I/II region binding site.