A simple and fast method was developed to determine non-UV active compounds directly without derivatization. The usefulness of the method was demonstrated by detecting the major components in aminoglycoside antibiotic mixtures using capillary zone electrophoresis with potential gradient detection. Under optimized separation conditions (0.2 mm cetyltrimethylammonium bromide (CTAB), 1 mm ammonium citrate, pH 3.5), gentamicin was separated into three major peaks (C1, C1a, and C2+C2a) within 15 min. This method showed better sensitivity than other capillary electrophoresis (CE) methods for determining underivatized gentamicin. The linear range was from 10 to 500 ppm. Because of its good repeatability and simplicity, this new method could be a good alternative for the current assays given by US Pharmacopoeia and European Pharmacopoeia.