AbstractActivity‐based proteomics (ABP) employs small molecular probes to specifically label sets of enzymes based on their shared catalytic mechanism. Given that the vast majority of lipases belong to the family of serine hydrolases and share a nucleophilic active‐site serine as part of a catalytic triad, activity‐based probes are ideal tools to study lipases and lipolysis. Moreover, the ability of ABP to highlight or isolate specific subproteomes results in a massive decrease of sample complexity. Thereby, in‐depth analysis of enzymes of interest with mass spectrometry becomes feasible. In this review, we cover probe design, technological developments, and applications of ABP of lipases, as well as give an overview of relevant identified proteins. © 2012 Wiley Periodicals, Inc. Mass Spec Rev 31:570–582, 2012