An efficient method of peptide thioester synthesis is described. The reaction is based on an N-4,5-dimethoxy-2-mercaptobenzyl (Dmmb) auxiliary-assisted N-S acyl shift reaction after assembling a peptide chain by Fmoc-solid phase peptide synthesis. The Dmmb-assisted N-S acyl shift reaction proceeded efficiently under mildly acidic conditions, and the peptide thioester was obtained by treating the resulting S-peptide with sodium 2-mercaptoethanesulfonate. No detectable epimerization of the amino acid residue adjacent to the thioester moiety in the case of Leu was found. The reactions were also amenable to the on-resin preparation of peptide thioesters. The utility was demonstrated by the synthesis of a 41-mer peptide thioester, a phosphorylated peptide thioester and a 33-mer peptide thioester containing a trimethylated lysine residue.