Endocannabinoids (ECs) are a unique group of lipids that function as chemical messengers in the nervous system. The two principle ECs thus far identified in mammals are N-arachidonoyl-ethanolamine (anandamide) and 2-arachidonoyl-glycerol (2-AG). These compounds have been implicated in various physiological and pathological functions including appetite, pain, sensation, memory, and addiction. Because ECs are synthesized and released on demand and then rapidly degraded to terminate signaling, the metabolic pathways that govern EC turnover directly influence the magnitude and duration of neuronal signaling events. There is strong evidence that two serine hydrolases, diacylglycerol lipase-alpha and -beta (DAGL-α and -β) function as 2-AG synthetic enzymes both in vitro and in vivo. However, because constitutive gene disruption, the only currently available means to investigate DAGL-α/β biology due to a lack of selective chemical inhibitors, can result in compensatory effects and network-wide changes, there is still uncertainty surrounding the extent to which DAGL-α/β contribute to 2-AG-mediated signaling. In an effort to provide chemical tools for manipulation of DAGL-β activity, we initiated a competitive activity-based protein profiling (ABPP) screen of triazole urea compounds to identify selective enzyme inhibitors. This campaign, made possible by previous inhibitor development efforts for LYPLA1/2 (ML211), PAFAH2 (ML225), and ABHD11 (ML226) based on the triazole urea scaffold, yielded the medchem optimized probe ML294 (SID 125269120). ML294 is highly potent against its target enzyme (IC50 = 56 nM in vitro; 12 nM in situ), and is active in vivo, showing both oral bioavailability and blood-brain barrier penetration. Out of more than 20 serine hydrolases (SHs) profiled by gel-based competitive ABPP, ML294 is observed to have one anti-target, alpha/beta hydrolase domain-containing protein 6 (ABHD6). Otherwise, ML294 is at least 35-fold selective for all other brain SHs (approximately 20) assessed by gel-based competitive ABPP and 7-fold selective vs. its closest homolog, DAGL-α. To control for ABHD6-directed activity in biological studies, we also developed a structurally related ABHD6-selective control “anti-probe”, ML295, also based on the triazole urea scaffold. The complete properties, characterization, and synthesis of ML294 are detailed in this report, and full details of ABHD6 inhibitors are detailed in the Probe Report for ML295 and ML296.