Enzyme inhibitors are widely screened for potency; however, selectivity is often tested only on lead candidates, resulting in many clinical failures. The authors developed EnPlex for multiplexed, high-throughput screening of compound potency and specificity. In EnPlex, small quantities of purified enzymes are coupled to colour-coded microbeads. Multiplexed bead–enzyme complexes are then incubated with an inhibitor that competes with activity-based fluorescent probes for binding to enzyme active sites. These mixtures are scanned by flow cytometry, in which one laser detects bead colour (enzyme identity) and another detects fluorescent signals (enzyme activity). EnPlex was validated by simultaneously screening >100 Ser hydrolases, a clinically relevant enzyme superfamily, against a panel of known inhibitors. The results confirmed known enzyme–inhibitor interactions and revealed hundreds of new interactions. EnPlex was then used to screen a library of electrophilic compounds, identifying lead molecules on the basis of both selectivity and potency. Unlike EnPlex, low-throughput methods can confirm enzyme–compound interactions in situ and do not require protein purification and immobilization; however, EnPlex offers both high-throughput coverage and increased sensitivity, and could be used to prioritize compounds during primary screening based on both potency and specificity.