A high-performance liquid chromatographic method for the quantitative determination of amikacin in serum is described. The antibiotic was separated from serum by adsorption on silica gel. The adsorbed drug was then derivatized with o-phthalaldehyde and eluted with ethanol. The derivatized amikacin was separated by reverse-phase, high-performance liquid chromatography and quantitated by fluorometry. A linear response for serum samples containing amikacin ranging from 1.0 to 15 mug/ml was obtained. Other antibiotics including various aminoglycosides did not interfere with the amikacin assay. Comparison with a standard microbiological assay gave a correlation coefficient of 0.99. This chemical assay is sensitive and specific under the conditions tested and can be performed rapidly.