A simple, rapid, and reproducible high-performance liquid chromatography method is described for the measurement of nafcillin using an internal standard (IS), 5-(p-hydroxyphenyl)-5-phenylhydantoin. A one-step protein precipitation was used for preparation of nafcillin and IS. The mobile phase included 0.02Mammonium acetate buffered solution and acetonitrile (75:25 vol/vol). An Ultrasphere ODS reversed-phase column was used and the detector was set at 224 nm. The retention time of IS and nafcillin was 4.8 and 6.6 min, respectively. The absolute recovery for nafcillin ranged from 95–98%. The intraday coefficient of variation for the assay was 3.5% at nafcillin concentrations of 1.0 and 60 mUg/ml. The interday coefficient of variation was 7.1 and 3.1% at the nafcillin concentrations of 1.0 and 60.0 mUg/ml, respectively. The major advantages of this method include the single-step extraction and the use of a small volume (50 mUl) of serum samples for nafcillin measurement. The serum concentrations of nafcillin in a pediatric patient were determined to demonstrate the application of this method.