Guanine nucleotide exchange factors (GEFs) are highly dynamic proteins and their function has been challenging to modulate with low molecular weight (LMW) compounds. GEFs activate distinct small GTPases relevant for human disease and hold promising therapeutic potential as novel drug targets. The first allosteric LMW inhibitors of VAV1 will be presented. Using biochemical screening and ligand-based hit expansion, we discovered a class of imidazopyrazines that potently and selectively inhibit VAV1-GEF dependent activation of RAC1. Initially, photo-affinity labeling was used to locate the binding site, which later was shown with high resolution co-crystal structures. The inhibitor-VAV1 complex is incompatible with binding to RAC1 leading to inhibition of guanine nucleotide exchange. Optimization led to a compound with an IC₅₀-value of 61 nM, a first in class allosteric VAV1 GEF inhibitor.