A liquid chromatographic method was developed and validated for the determination of erythromycin an oxime and related substances. The chromatographic analysis was performed on Inertsil C18 ODS column, using the phosphate buffer solution (0.02 M, pH 6.5)-acetonitrile (40:60, V/V) as the mobile phase. Wavelength of the UV detector was 215 nm. All four related substances, including Z isomer of erythromycin A oxime, erythromycin A, erythromycin A-6, 9-hemiketal and erythromycin A-6, 9-9, 12-spiroketal were separated and determined under isocratic elution. The effect of pH and the concentration of mobile phase on separation were studied. The developed method was successfully applied to the analysis of erythromycin an oxime in commercial formulation.