A simple and sensitive HPLC method was developed for the quantitative analysis of nargenicin in the culture broth of Nocardia sp. CS682, isolated from Korean soil. Nargenicin, an unusual macrolide antibiotic with potent anti-MRSA (methicilin-resistant Staphylococcus aureus) activity, was extracted from the culture broth with ethylacetate and then analyzed by HPLC using solvent A (0.5 M dipotassium phosphate pH 2.5-water: acetonitrile = 80:20, v/v) and solvent B (100% acetonitrile), at 215 nm. The calibration curve of nargenicin was linear with correlation coefficient greater than 0.99. This method provides precise analysis of the nargenicin amount present in the cell free culture medium, and may be applicable to analyze other bacterial cultures containing similar compounds.