The native chemical ligation enables the chemoselective coupling of two unprotected protein segments. In the initial format of this reaction, the side chain of N-terminal cysteine residues serves to capture a peptide thioester. The N-terminus of the cysteinyl peptide is involved in a subsequent intramolecular aminolysis reaction, upon which the “native” peptide bond is established. Considerable efforts have been invested to remove the restriction to cysteine-containing ligation sites. In this review, we focus on the combination of two chemoselective reactions, native chemical ligation, and desulfurization, which has extended the repertoire of accessible ligation junctions. Based on this two-step approach, native alanine-containing peptides are accessible through postligation desulfurization of cysteine-ligation products. Recently, significant progress has been achieved in the development of new thiolated building blocks that serve as precursors to other amino acids. Ligations at phenylalanine can be accomplished by means of a β-mercaptophenylalanine building block and subsequent desulfurization. The building blocks β-mercaptovaline (penicillamine) and γ-mercaptovaline provide access to hydrophobic ligation sites (Xaa-Val). Lysine has been equipped with a δ- and γ-mercapto groups, which enables ligations at both the α- and the ε-amino group. This review also describes the recent improvements of desulfurization chemistry, which have widened the scope of the ligation−desulfurization approach and offer a promising alternative for the synthesis of Cys-free peptides and glycopeptides. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 551–559, 2010.