The compounds were serially diluted in DMSO to result in concentrations covering the compound activity range (usually between 12.5 µM and 70 pM). The dilutions were transferred to a 384-well assay plate containing a recombinant MAGL protein in assay buffer (50 mM TRIS, 1 mM EDTA, 0.01% Tween 20, v/v and 2.3% DMSO, v/v) and incubated at room temperature for 15 min. The reaction was initiated by adding the substrate 2-AG in assay buffer, and the incubation was conducted with gentle shaking at room temperature for 30 min. The final concentration of the MAGL protein was 50 pM, 8 µM for 2-AG, and 2.5% (v/v) DMSO. Upon the completion of incubation, the reactions were quenched with a double assay volume of acetonitrile containing 4 µM d8-arachidonic acid (AA). The build-up of d8-AA was monitored using an online solid-phase extraction system (Agilent Rapid-Fire) coupled to a triple quadrupole mass spectrometer (Sciex5000 or Agilent 6460). The samples were loaded on a C18 cartridge with 99% water/acetonitrile (v/v) and eluted with 5 mM ammonium acetate in 90% acetonitrile (v/v). A mass spectrometer was operated in the ESI mode with the mass transitions m/z = 303.1-259.1 for AA and m/z = 311.1-267.1 for d8-AA, respectively. The IC 50 values were fitted based on the ratio of intensities of AA/d8-AA at the different assay points of the serial dilution.