A spectrophotometric assay method for the quantitative determination of amikacin, kanamycin, neomycin, and tobramycin in pharmaceutical dosage forms has been developed. The method is based on the Hantzsch reaction, forming dihydrolutidine derivatives which can be measured spectrophotometrically. The excipients EDTA, phenol, sodium bisulfite, and sodium citrate do not interfere, while salts of ammonia do interfere. The relative standard deviations based on seven readings were 1.64, 1.88, 2.10, and 1.93% for amikacin, kanamycin, neomycin, and tobramycin, respectively. Assay results have been compared with microbiological assay results provided by the manufacturers. The assay method appears to be stability indicating.