3-Amino-2-keto-7H-thieno[2,3-b]pyridin-6-one derivatives were discovered as moderately potent inhibitors of ubiquitin C-terminal hydrolase-L1 (UCH-L1) utilizing an assay that measures hydrolysis of the fluorogenic substrate Ub-AMC. SAR studies revealed that both the carboxylate at the 5-position and the 6-pyridone ring were critical for inhibitory activity. Furthermore, activity was dependent on the nature of the ketone substituent at the 2-position, with 4-Me-Ph and 2-naphthyl being best. Kinetic mechanism studies revealed that these compounds were uncompetitive inhibitors of UCH-L1, binding only to the Michaelis-complex and not to free enzyme. The active compounds were selective for UCH-L1, exhibiting neither inhibition of other cysteine hydrolases (e.g., UCH-L3, papain, isopeptidase T, caspase-3, and tissue transglutaminase) nor cytotoxicity in N2A cells.