Boronic acids (BAs) are a prominent functionality extensively used to design biologically active compounds and functional biomaterials. Boronic acids open shell can lead to unspecific reactivity of BAs with endogenous nucleophiles and to undesired off-target effects. Here, diazaborines are presented as a new class of boron-based warheads for serine proteases inhibition, in which the boron function is stabilized in the form of an aromatic boron-based heterocycle. In this study, diazaborines were readily synthesized in a single step in yields up to 96%, without any chromatographic operation and were shown to reversibly inhibit human neutrophil elastase (HNE) serine protease with IC50 values in the low μM range. Diazaborines were also selective toward HNE, as no inhibition was observed against a panel of five closely related serine proteases. Synthetic and theoretical studies performed on this system suggest that, similar to BAs, the reaction mechanism involves the formation of a reversible covalent bond between the diazaborine boron center and the catalytic serine oxygen. Finally, different from BAs, diazaborines were shown to be very stable in pH 7.4 phosphate buffer and human plasma.