The protocol described in this chapter details the synthesis of Desotamides, a class of cyclic hexapeptide antibiotics from Streptomyces species. These peptides, which often include D-amino acids and non-proteogenic amino acids, are challenging to produce using molecular biology techniques. Their synthesis involves loading the first amino acid onto 2-chlorotrityl chloride resin, followed by fluorenylmethyloxycarbonyl (Fmoc) solid phase synthesis of the linear peptide, cleavage of a protected peptide fragment from the resin using mild acidic conditions, and head-to-tail cyclization in solution. These synthetic processes permit the scalable production and engineering of Desotamides and novel-related antimicrobial species, without the limitations of biological synthesis methods.