A method for the determination of impurities and degradation products in nalbuphine hydrochloride injection by gradient elution high performance liquid chromatography (HPLC) is reported. Reversed phase gradient elution chromatography was carried out using a mobile phase containing 0.05% trifluoroacetic acid, acetonitrile, and tetrahydrofuran. Validation data for linearity, accuracy, precision, robustness, detection limit, and quantitation limit are presented. The chromatographic system resolves nalbuphine from related substances with corrections made for differences in detector response at the specified wavelength.