A reversed-phase high performance liquid chromatographic mothod was developed for the determination of berberine in Cortex Phellodendri using a bonded phase column (Zorbax C8) with diazepam as the internal standard. The column temperature was 25 C. The mobile phase was a mixture of acetonitrile-water (35:65, containing 30mmol/L H3PO4) with a flow rate of lml/min. Berberine was detected by UV absorption at 346nm. The standard curve was linear in the range of 0.14 to 2.0 micrograms. The recovery was 101.7% and RSD 1.83%.