A rapid high performance liquid chromatography for determination of demethylvancomycin in neonate serum has been developed. The procedure involved a simple protein precipitation by acetonitrile-isopropanol (1:1) and then the sample was chromatographed on a reversed phase C18 column with UV detection at 236 nm. The mobile phase was CH3CN: 0.05 mol/L KH2PO4 = 8:92 (V/V). The calibration curve was Y = 35,721.89X - 13,031.54, r = 0.9998 and he detection limit was 0.3 mg/L. The average recovery was 94.7% +/- 1.2%. Intra-day and inter-day RSD were 2.23% and 2.62% respectively. It can be concluded that this method meets the requirement for routine clinical application. This method has been used to determine serum concentration of demethylvancomycin in neonates. The data obtained showed that the method was simple, rapid, sensitive and precise.