Monoacylglycerol lipase (MAGL) is a membrane-associated cytosolic serine hydrolase which catalyses the hydrolysis of the endocannabinoid 2-arachidonoylglycerol into arachidonic acid and glycerol. MAGL represents the link between the endocannabinoid and the eicosanoid system indeed its inhibition enhances endocannabinoid signalling and lowers eicosanoid production. Here we present a radioactive-free, sensitive and solid HPLC–UV based method to evaluate MAGL activity by using 4-nitrophenylacetate (4-NPA) as substrate. The enzymatic activity is measured by quantifying the 4-nitrophenol (PNP) (λ = 315 nm) formation on a C18 stationary phase. The method was validated by calculating IC50 values of the reference inhibitors JZL184, CAY10499 and JW642 and confirming the irreversible and non-competitive mechanism of inhibition for JZL184. Furthermore in order to resemble the catalytic conditions of MAGL at cell membrane level, the surfactant Triton X-100 was added, as a micelle forming agent and 4-nitrophenyldodecanoate (4-NPDo) was used as lipophilic substrate for MAGL. The data obtained confirmed that the HPLC method is an alternative, radioactive-free approach for the screening and characterization of new MAGL inhibitors. Finally this assay prevents, in an unequivocal manner, any interference related to the intrinsic absorbance of screened compounds or metabolites generated upon enzymatic cleavage which could seriously affect the assay readout.