Sparsomycin, an antitumor agent and inhibitor of protein synthesis with an unusual chiral sulfur moiety, has been the subject of studies in structure-activity relationships, mechanism of action, and activity potentiation. The Chemotherapy Fermentation Laboratory was assigned to produce pure sparsomycin for the National Cancer Institute (NCI), but scale-up from shake flasks (titers up to 180 pg/ml) to 30-gallon fermentors led to low titers (<30 pg/ml). A fast assay was critical to harvest at peak titer, as prolonged fermentation produced tubercidin, which interfered with sparsomycin recovery. The previous normal phase high-performance liquid chromatography (HPLC) assay was unsuitable for production due to slow sample work-up and incompatible mobile phase. A reversed-phase HPLC assay requiring minimal sample preparation (mobile phase: 0.05 M potassium phosphate buffer pH 7.8-methanol 85:15) was developed. This method was much faster, simpler, and cost-effective than the prior one. It supported fermentation optimization, increasing titers to ~100 pg/ml, improved recovery yield, and enabled delivery of 10 g of 99% pure sparsomycin to the NCI. Additionally, the assay could resolve 9 out of 11 nucleoside antibiotics and identify toyocamycin in an unknown fermentation broth.