In chemical protein synthesis, peptide building blocks are prepared by solid-phase peptide synthesis (SPPS), and then connected by chemical ligation methods. The peptide thioester is one of key building blocks used in chemical protein synthesis, and improvements in the Fmoc SPPS procedure for preparing such thioesters would be highly desirable. In this review we focus on a method for peptide thioester synthesis based on the use of an intramolecular N to S acyl shift reaction as a key reaction. Amide and thioester forms at the thiol-containing residue are in equilibrium as a result of a reversible intramolecular acyl shift, which is detectable by 13C NMR. The amide form is favored under neutral conditions, while the thioester predominates under acidic conditions. Thiol auxiliaries can be employed to facilitate the formation of a thioester from an amide via an intramolecular N-S acyl shift, and the peptide thioester is formed after intermolecular transthioesterification in the presence of excess amounts of thiols. Even under neutral conditions, thiol auxiliary-containing peptides can be ligated with a cysteinyl peptide via an intramolecular N-S acyl shift, followed by native chemical ligation (NCL) in a one-pot reaction. These procedures can be applied to the chemical synthesis of proteins which are post-translationally modified.