we report a strategy for creating mechanism-based, light activated protease and hydrolase substrate traps in complex mixtures and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid permits the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond; this enables stringent purification and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4. We demonstrate that RHBDL4 can shed luminal fragments of ER-resident type I transmembrane proteins to the extracellular space, as well as catalysing non-canonical secretion of endogenous soluble ER-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 is an aminopeptidase – with a preference for removing aromatic amino acids – in human cells. Our results exemplify a powerful paradigm for discovering the substrates and activities of hydrolase enzymes.