A stability-indicating normal-phase high-performance liquid chromatographic (HPLC) method for the assay of novobiocin has been developed. The method uses a silica column with a mobile phase composed of butyl chloride-tetrahydrofuran-methanol-acetic acid (88:5:4:3). The amount of acetic acid in the mobile phase significantly influences the resoltuion of novobiocin and desmethyldescarbamylnovobiocin peaks. Isonovobiocin and degradation compounds of novobiocin have been separated. The standard curve for the quantification of novobiocin was linear in the range of 0.2–0.6 mg novobiocin per ml and the relative standard deviation of the assay was less than 1%. Excellent correlation was obtained between the HPLC and microbiological assay methods. The method was used to assay novobiocin in mastitis products