Significance Extracellular signal-regulated kinase (ERK) is a critical enzyme that interacts with a wide range of regulators and substrates to control cellular functions. ERK interactions have been studied using a number of biophysical and biochemical techniques, which identified two docking domains on ERK used by almost all of its substrates and regulators. However, mapping the binding interface of a new ERK interactor with little prior knowledge of the binding mechanism remains a challenge. Here we show that the introduction of a photocrosslinking amino acid into the heterodimerization interface of ERK and its substrate, Capicua, allows for precise, amino-acid–level determination of the substrate binding site using tandem mass spectrometry. This study demonstrates the utility of photocrosslinking approaches for the determination of binding sites on kinases.