An RP-HPLC method with photodiode array detection has been developed for the determination of major constituent berberine from Berberis aristata (Barberry) and Berberis tinctoria. Berberine was isolated from the plant extract on semi-preparative HPLC and separated on HPLC by using an isocratic mode consisting of 0.1% trifloroacetic acid: acetonitrile (60:40， v/v) at a flow rate of 1 mL/min. Under these conditions’ a plot of integrated peak area versus concentration of berberine was found be linear over the concentration range of 0.2 |ig/mL to 150 |ig/mL. The limit of detection was 1ng on column and limit of quantification was 2 ng on column for berberine. The berberine content in B. aristata and B. tinctoria was found to be 3.18% and 1.46% respectively.