<p>Reprogramming the specificity of proteases toward alternative target sequences could enable an array of exciting applications, ranging from proteome editing to therapeutic interventions. Here we report an in vivo life–death selection system for protease reprogramming using the toxic N-terminal domain of gasdermin D (GD-N) protein as a selection marker. The approach is a modular system that can be used to cover the protease mutational diversity in the billions through only a few cycles of directed evolution. By inserting the desired cleavage sequence into the loop region of the GD-N protein, which is toxic to host bacteria cells, the system selects for efficient substrate cleavage—rendering GD-N nontoxic—by enrichment of bacteria in liquid culture. Using the tobacco etch virus protease (TEVp) and corresponding substrate sequence as a model, we demonstrated that our platform could select and enrich an efficient protease variant millionfold after a single round of selection. We also evolve TEVp to cut sequences on target proteins with known pathological roles.</p><p></p>