A total synthesis of daptomycin, the first natural product antibiotic launched in a generation, was achieved. This convergent synthesis relies on an efficient macrocyclization via a serine ligation to assemble the 31-membered cyclic depsipeptide. The difficult esterification by the nonproteinogenic amino acid kynurenine was accomplished via the esterification of a threonine residue by a suitably protected Trp ester, followed by ozonolysis. This synthesis provides a foundation and framework to prepare varied analogues of daptomycin to establish its structure-activity profile.