The selectivity of a chemical probe or drug is arguably more important than its potency, yet selectivity is rarely assessed comprehensively. The explanation for this is simple: testing for specificity is too difficult and expensive with the current state of the art technologies, especially in the early stages of drug discovery. Here, I will describe EnPlex, an efficient, high-throughput method for simultaneously assessing inhibitor potency and specificity, and its pilot application to >100 serine hydrolases. I will highlight several notable examples in which EnPlex streamlined inhibitor discovery by enabling specificity information to help drive lead candidate selection and optimization efforts. Moreover, I will describe how EnPlex analysis of previously described serine hydrolase inhibitors has revealed numerous unrecognized off-target interactions, some of which may help explain previously confounding biological activities. In particular, I will discuss how EnPlex selectivity profiling helped elucidate the mechanism of action of the immune-stimulating small molecule Val-boroPro.