This account reports on the development and function of novel substrate mimetics as artificial substrates for Glu-specific endopeptidases. Firstly, in an empirical way, various aliphatic and aromatic analogs of the already established carboxymethyl thioester-substrate mimetics were designed from simple structure-function relationship studies. The specificity of the newly developed substrates for Staphylococcus aureus V8 protease-catalyzed reactions have been examined by steady-state hydrolysis kinetic studies. Additionally, these studies were expanded to the use of the equally Glu-specific endopeptidase from Bacillus licheniformis (BL-GSE) which can easily be purified from alcalase in high yields. Finally, the novel substrate mimetics were used as acyl donor components in BL-GSE- and V8 protease-catalyzed model acyl transfer reactions. The results clarify the newly developed substrate mimetics as efficient acyl donors as well as BL-GSE as an attractive alternative to V8 protease for enzymatic peptide synthesis.