Abstract A fast, selective, and precise liquid chromatographic method for simultaneous, independent determination of kanamycins A and B is described. Sample components are separated on a pellicular cation exchanger and monitored by fluorescence using post-column on-line derivatization. Less than 0.35 μg of kanamycin B can be detected in as much as 7 μg kanamycin A injected. The detection limit for kanamycin A is less than 20 ng injected. Reproducibility of the entire chromatographic system is about 1% (2 σ) based upon repeated injections of standards. Precision of repeated process sample preparations is about 6% (2 σ). Chromatographic analysis time is less than 15 min per sample.