Semisynthesis using recombinant polypeptides as building blocks is a powerful approach for the preparation of proteins with a variety of modifications such as glycosylation. The activation of the C terminus of recombinant peptides is a key step for coupling peptide building blocks and preparing a full-length polypeptide of a target protein. This article reports two chemical approaches for transformation of the C terminus of recombinant polypeptides to thioester surrogates. The first approach relies on efficient substitution of the C-terminal Cys residue with bis(2-sulfanylethyl)amine (SEA) to yield peptide-thioester surrogates. The second approach employs a native tripeptide, cysteinyl-glycyl-cysteine (CGC), to yield peptide-thioesters via a process mediated by a thioester surrogate. Both chemical transformation methods employ native peptide sequences and were thereby successfully applied to recombinant polypeptides. As a consequence, we succeeded in the semisynthesis of a glycosylated form of inducible T cell costimulator (ICOS) for the first time.