It is difficult to determine a chemical inhibitor's binding site in multiprotein mixtures, particularly when high-resolution structural studies are not straightforward. Building upon previous research involving photo-cross-linking and the use of mixtures of stable isotopes, we report a method, Stable Isotope Labeled Inhibitors for Cross-linking (SILIC), for mapping a small molecule inhibitor's binding site in its target protein. In SILIC, structure-activity relationship data is used to design inhibitor analogues that incorporate a photo-cross-linking group along with either natural or 'heavy' stable isotopes. An equimolar mixture of these inhibitor analogues is cross-linked to the target protein to yield a robust signature for identifying inhibitor-modified peptide fragments in complex mass spectrometry data. As a proof of concept, we applied this approach to an ATP-competitive inhibitor of kinesin-5, a widely conserved motor protein required for cell division and an anticancer drug target. This analysis, along with mutagenesis studies, suggests that the inhibitor binds at an allosteric site in the motor protein.