Tandem ligation for the synthesis and modification of proteins entails forming two or more regiospecific amide bonds of multiple free peptide segments without-a protecting-group scheme. We here describe a semi-orthogonal strategy for ligating three unprotected peptide segments, two of which contain N-terminal (NT) cysteine, to form in tandem two amide bonds, an Xaa-SPro (thiaproline), and then an Xaa-Cys. This strategy exploits the strong preference of an NT-cysteinyl peptide under acidic conditions to undergo selectively an SPro-imine ligation rather than a Cys-thioester ligation. Operationally, it was performed in the N --> C direction, first by-an imine ligation at pH < 3 to afford an Xaa-thiazolidine ester bond between a: peptide containing a carboxyl terminal (CT)-glycoaldehyde ester and a second peptide containing both-an NT-Cys and a CT-thioester. The newly created O-ester-linked segment with a CT-thioester was then ligated to another NT-cysteinyl peptide through thioester ligation at pH > 7 to form an Xaa-Cys bond. Concurrently, this basic condition also catalyzed the O,N-acyl migration of an Xaa-thiazolidine ester to the Xaa-SPro bond at the first ligation site to complete the tandem three-segment ligation. Both ligation reactions were performed in aqueous buffered solvents. The effectiveness of this three-segment ligation strategy was tested in six peptides ranging from 19 to 70 amino acids, including thiaproline --> proline analogues of somatostatins and two CC-chemokines. The thiaproline replacements in these peptides and proteins did not result in altered biological activity. By eliminating the protecting-group scheme and coupling reagents, tandem ligation of multiple free peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach for combinatorial segment synthesis.