ARN14686 is an activity-based affinity probe for the detection of N-acylethanolamine acid amidase (NAAA) using click chemistry. It binds covalently to the N-terminal cysteine of catalytically active NAAA to form a thioester adduct. ARN14686 inhibits the hydrolysis of the NAAA substrate PAMCA in HEK293 cells (ICs = 6 and 13 nM for human and rat recombinant enzymes, respectively) and is selective for NAAA over acid ceramidase (IC = 1,200 nM for the rat enzyme). It labels NAAA in HEK293 cells overexpressing human or rat recombinant NAAA and, when used at concentrations of 1 and 10 µM, in rat lung lysosomal fractions in vitro. It also detects NAAA in lung lysosomal fractions following intravenous administration at a dose of 10 mg/kg in rats. ARN14686 (3 mg/kg) has been used to determine that catalytically active NAAA is present in inflamed rat paw in a complete Freund’s adjuvant model of inflammation.