ML349 is a reversible APT2 inhibitor with Ki = 120 nM. ML349 is highly selective within the serine hydrolase enzyme family, it could still interact with other cellular targets. In human cell lysates, biotinylated-ML349 enriches a recurring set of proteins, including metabolite kinases and flavin-dependent oxidoreductases that are potentially enhanced by avidity-driven multimeric interactions. ML349 achieves target engagement and hydrolase selectivity in living mice.