Over 100 protease inhibitors are currently used in the clinics, and most of them use blockage of the active site for their mode of inhibition. Among the protease drug targets are several enzymes for which the correct multimeric assembly is crucial to their activity, such as the proteasome and the HIV protease. Here, we present a novel mechanism of protease inhibition that relies on active-site-directed small molecules that disassemble the protease complex. We show the applicability of this mechanism within the ClpP protease family, whose members are tetradecameric serine proteases and serve as regulators of several cellular processes, including homeostasis and virulence. Compound binding to ClpP in a substoichiometric fashion triggers the formation of completely inactive heptamers. Moreover, we report the selective β-sultam-induced dehydroalanine formation of the active site serine. This reaction proceeds through sulfonylation and subsequent elimination, thereby obliterating the catalytic charge relay system. The identity of the dehydroalanine was confirmed by mass spectrometry and crystallography. Activity-based protein profiling experiments suggest the formation of a dehydroalanine moiety in living S. aureus cells upon β-sultam treatment. Collectively, these findings extend our view on multicomponent protease inhibition that until now has mainly relied on blockage of the active site or occupation of a regulatory allosteric site.