Bacitracin, a polypeptide antibiotic, is one of the most commonly used antibiotics in the world. The approved method of analysis for bacitracin is microbial. To correlate the microbiological method with a high-performance liquid chromatographic (HPLC) method, bacitracin was chromatographed using HPLC with ultraviolet detection and a YMC basic column. Adequate separation of the isomers was obtained to scale up this procedure to preparative HPLC using a Prep HPLC system and a 250 x 21 mm YMC basic column. The various fractions were separated, isolated and examined for microbial activity. The individual fractions could be precipitated by adding zinc or methylene disalicylic acid and lowering the pH. The crude fractions were recycled to ensure chromatographic purity. The chromatograms can accurately predict (in minutes) the microbiologically determined potency which usually takes 16-24 h to develop. The chromatographic procedure also provides information on the amounts of isomers and degradation products present in the sample, whereas the microbiological assay only provides activities or potencies of the antibiotic. The reported HPLC method also possesses some advantages over some other published HPLC methods in terms of accuracy and time of analysis.