The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly biosynthesized by sn-1-diacylglycerol lipase alpha (DAGL-alpha) in the CNS. Selective inhibitors of DAGL-alpha will provide valuable insights in the role of 2-AG in endocannabinoid signaling processes and are potential therapeutics for the treatment of obesity and neurodegenerative diseases. Here, we describe the development of a natural substrate-based fluorescence assay for DAGL-alpha, using a coupled enzyme approach. The continuous setup of our assay allows monitoring of DAGL-alpha activity in real-time and in a 96-well plate format. This constitutes a major improvement to the currently available radiometric and LC/MS-based methods, which can be executed only in low-throughput formats. In addition, our assay circumvents the use of radioactive material. We demonstrate that our assay can be used to screen inhibitors of DAGL-alpha activity, using 1-stearoyl-2-arachidonoyl-sn-glycerol as the physiologically relevant natural substrate of DAGL-alpha. Furthermore, our method can be employed to measure DAGL activity and inhibition in the mouse brain membrane proteome. Consequently, our assay should serve as a valuable tool for rapid hit validation and lead optimization of DAGL-alpha inhibitors.