The synthesis and development of N-terminal peptidic boronic acids as protease inhibitors is reported. N-Terminal peptidic boronic acids interrogate the S′ sites of the target protein for selectivity and provide a new strategy that complements the currently known peptidic α-amino boronic acids (C-terminal boronic acids). After screening a series of N-terminal peptidic boronic acids, the first selective inhibitor of human ClpXP, an ATP-dependent serine protease present in the mitochondrial matrix, was discovered. This should facilitate the understanding of the physiological function of this protease