Fourteen gentamicin sulfate lots collected from international markets showed high quantities of impurities (approximately 30% of studied lots). 1H NMR spectroscopy as a primary analytical method was applied in order to validate the quantification results obtained from micellar electrokinetic chromatography method (MEKC). In this study, 1H NMR data of 46 gentamicin sulfate drug substance lots were used to classify the lots by means of principal component analysis (PCA) of 14 1H NMR-signals in the 5.0-6.0 ppm region. Three main groups could be classified: high purity (3 lots), average quality (28 lots) and low purity (14 lots); one lot proved to be atypical. The 14 normalized signal heights in the 5.0-6.0 ppm region are predictive for purity quality according to a partial least squares (PLS)-model with sum of all impurities as Y-variable (measured by MEKC).