Monoacylglycerol lipase (MGL) is a serine hydrolase that regulates endocannabinoid signaling. Overexpression of this enzyme in certain tumor cells elevates the level of protumorigenic signaling lipids, and as such, MGL participates in a fatty acid network that promotes cancer pathogenesis. MGL is the major enzyme responsible for the hydrolysis of 2-arachidonoylglycerol (2-AG) — increased levels of which are considered antinociceptive, antiallodynic, anti-inflammatory and therapeutic for neurodegenerative disorders. Also, MGL inhibition has been shown to attenuate the growth, migration and invasion of aggressive/metastatic, prostate and breast cancer cells. The development of a highly selective imaging modality for examining functional MGL will be a key step in our understanding of the complete metabolic role this potential biomarker for breast cancer plays. AM3506 is a sulfonyl fluoride-based, potent and in vivo effective dual MGL and fatty acid amide hydrolase (FAAH) inhibitor. Here we show, using liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-QTOF MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), that AM3506-based enzyme inhibition occurs through sulfonation of the catalytic serine, followed by beta elimination (b/e) in MGL, but not in FAAH. We also show that His128, which precedes the catalytic Ser129 in MGL, is essential for this b/e which converts this serine into an alpha-beta unsaturated amide; an ideal Michael acceptor. Here, we used activated thiols as Michael donors under physiological conditions to specifically label recombinant human MGL. This proof-of-concept experiment opens the door for the development of novel, fluorogenic probes that target catalytically active human MGL in vivo.