BACKGROUND Cathepsin G (CatG) is a cationic serine protease with a wide substrate specificity. CatG has been reported to play a role in several pathologies, including rheumatoid arthritis, ischemic reperfusion injury, acute respiratory distress syndrome, and cystic fibrosis, among others. OBJECTIVE We aim to develop a new class of CatG inhibitors and evaluate their potency and selectivity against a series of serine proteases. METHODS We exploited chemical synthesis as well as chromogenic substrate hydrolysis assays to construct and evaluate the new inhibitors. RESULTS In this communication, we report on a new class of CatG inhibitors of 4H-3,1-benzoxazin- 4-one derivatives. We constructed a small library of seven substituted 4H-3,1-benzoxazin-4-one derivatives and identified their inhibition potential against CatG. Five molecules were identified as CatG inhibitors with values of 0.84-5.5 μM. Inhibitor 2 was the most potent, with an IC50 of 0.84 ± 0.11 μM and significant selectivity over representative serine proteases of thrombin, factor XIa, factor XIIa, and kallikrein. CONCLUSIONS Thus, we propose this inhibitor as a lead molecule to guide subsequent efforts to develop clinically relevant potent and selective CatG inhibitors for use as anti-inflammatory agents.