AbstractSerine hydrolases (SHs) comprise a large superfamily of enzymes that play critical roles in many biological processes. Despite their importance, many SHs remain uncharacterized and the vast majority of SHs lack selective inhibitors. In response, activity‐based protein profiling (ABPP) and activity‐based probes (ABPs) have been leveraged to construct a more comprehensive picture of the SH proteome. Since the utility of ABPP is largely dictated by the reactivity profile of the ABPs deployed, novel scaffolds and chemotypes are needed to expand the breadth and selectivity of SH‐targeting ABPs. In this review, we highlight recent innovations in SH probe development, covering both established and emerging electrophilic warheads. We then discuss how strategic implementation of SH‐targeting ABPs has yielded selective, potent inhibitors and imaging agents with broad use. Finally, we present methods for ABP diversification and explore cutting‐edge applications in therapeutics development and discovery biology.