Methyl nonactate is a valuable starting material for the production of natural product-like combinatorial libraries and ketide amino acids, a series of novel conformationally constrained amino acid analogues. Fermentation of Streptomyces griseus generates high titers of macrotetrolide antibiotics from which methyl nonactate can be generated by methanolysis. Unfortunately, this approach generates mixtures of homologues that are not economically separable. We report a specific lipase-mediated hydrolysis of nonactate derivatives that discriminates between homologues and is the foundation of an economically tractable preparation of methyl nonactate in scale.