Abstract2‐Arachidonoylglycerol (2‐AG) is an important endogenous signaling lipid that activates the cannabinoid receptors (CB1R and CB2R), thereby regulating a diverse range of physiological processes including anxiety, appetite, inflammation, memory, pain sensation, and nociception. Diacylglycerol lipases (DAGLs) are the principle enzymes responsible for 2‐AG biosynthesis. Recently, the (patho)physiological functions of DAGLs have been explored by both genetic methods and chemical tools. This review will focus on the recent efforts to develop highly selective and in vivo active DAGLs inhibitors using activity‐based protein profiling.