We report an Fmoc-compatible and external-thiol-free method of peptide C-terminus thioesterification with cysteinylprolyl imide. The newly synthesized structure, i.e., cysteinylprolyl-thiazolidinone, provided high conversion and sequence-independent fast kinetics (90 min) in the diketopiperazine thioester formation under relatively mild conditions: pH 6.0, 37 °C. Employing this thioesterification method, we synthesized histone H3.2 bearing K56 acetylation.