AbstractA rapid reversed‐phase high‐performance liquid chromatographic (rp‐HPLC) assay for the determination of baicalin in rat thalamus was developed. This was carried out on a Hypersil –C18 column using 4‐nitro‐benzoic acid as the internal standard with a mobile phase of methanol–water–H3PO4 (45:55:0.2, v/v/v). Detection was by UV at 277 nm. The calibration curve for baicalin was linear (r = 0.9992) over the concentration range of 0.05–4.0 µg/mL and the limit of detection was 10 ng/mL. The coefficients of variation of intra‐ and inter‐day assays were 2.64, 5.19 and 3.19% and 3.46, 6.21 and 5.58% at concentrations of 0.5, 1.0 and 3.0 µg/mL, respectively. The recoveries of baicalin from rat thalamus were 85.4 ± 5.62, 90.7 ± 2.43 and 89.1 ± 4.75% at concentrations of 0.5, 1.0 and 3.0 µg/mL, respectively. The method was applied to determine the time course of baicalin in rat thalamus, following a single dosage of intravenous administration of Scutellariae radix extract at 90 mg/kg of baicalin to male Wistar rats. Copyright © 2005 John Wiley & Sons, Ltd.