In this contribution we describe a general synthesis concept for the in situ preparation of protease specific reactants using methyl thioesters as universal precursors. The precursor esters are readily available by standard synthesis procedures and can be used directly as reactants for protease-mediated peptide coupling reactions. Alternatively, they can serve as initial building blocks for the in situ preparation of various types of substrate mimetics. The synthesis of the latter is achieved by a one-pot spontaneous transthioesterification reaction of the parent thioester (Y-(Xaa)(n)-SMe-->Y-(Xaa)(n)-SR; R: CH2CH2COOH, CH2C6H5, C6H4NHC(:NH)NH2), which proceeds efficiently in both a sequential manner and parallel to the subsequent enzymatic reaction. The resulting substrate mimetics act as efficient acyl donor components and show the typical behavior of substrate mimicry enabling irreversible reactions with originally nonspecific acyl moieties. Neither a workup of the substrate mimetic intermediate nor changes of the reaction conditions during the whole synthesis process are required. Model peptide syntheses using trypsin, alpha-chymotrypsin, and V8 protease as the biocatalysts proved the function of the approach and illustrated its synthetic value for protease-mediated reactions and the compatibility of the approach with state-of-the-art solid-phase peptide ester synthesis methods.