Measurement of serum tobramycin and gentamicin by high-performance liquid chromatography (HPLC) was evaluated with the purpose to apply to the routine practice of medicine. The method is based on the technique reported by Anhalt but is slightly modified to achieve our purpose. Analytical procedure by HPLC includes:(1) extraction from serum using CM-Sephadex®,(2) separation of an extract by ion-paired chromatography,(3) derivation by post-labeled method. The time required to one assay is less than 30 minutes. The maximum within-day and between-day variance (CV %) were 2.9 and 6.2% respectively for measurement of gentamicin. The same degree of precision (4.0 %) and reproducibility (4.2%) were observed in tobramycin assay. The recovery ratio ranged from 95.4 to 105.6% in gentamicin and from 97.1 to 103.8% in tobramycin assay. The HPLC assay correlated well with bioassay, radioimmunoassay and enzymeimmunoassay for measuring serum concentration of tobramycin in sera from healthy subjects and patients.(n=142 or 154, r=0.92-0.98) The method may prove more suitable for aminoglycoside drug monitoring than other methods and conventional HPLC technique, as the technique employed in this study is rapid, cost-effective and appropriate to measure a single sample, if necessary.