A novel HPLC-UV method was developed for the simultaneous determination of two major active components in Yinhuang injection, chlorogenic acid and baicalin, in rat plasma. Extracted from the plasma samples with methanol-acetonitrile (3:1, v/v), the two compounds were successfully separated using a C18 column with a gradient elution composed of 15 and 54% methanol-acetonitrile (1:1, v/v) in 0.2% (v/v) phosphoric acid water solution (pH 2.0). The flow-rate was set at 1 ml min(-1) and the eluent was detected at 327 nm for chlorogenic acid, 278 nm for baicalin. Puerarin and rutin were used as the internal standards for chlorogenic acid and baicalin, respectively. The method was linear over the range of 0.388-12.4 microg ml(-1), 0.485-124 microg ml(-1) for chlorogenic acid and baicalin, respectively. The correlation coefficient for each analyte was above 0.998. The intra-day and inter-day precisions were better than 7 and 9%, with the relative error ranging from -9.5 to 7.3% and from -4.2 to 1.8%. The limit of detection (LOD) and the limit of quantification (LOQ) for chlorogenic acid and baicalin in plasma were 0.194, 0.122, 0.388 and 0.485 microg ml(-1), respectively. This assay has been successfully applied in the pharmacokinetic study of chlorogenic acid and baicalin in vivo through intravenous administration of Yinhuang injection to rats.