Berberis vulgaris L. belongs to family Berberidaceae is native to Europe and the British Isles in Iran. Barberries is an important production of South Khorasan; biggest producer of barberries in Iran. It is a deciduous shrub having yellow flowers and scarlet colored fruit in the form of berries. Twenty two alkaloids have been reported so far from root, stem leaves and fruit of this plant, which are of medicinal importance. From preliminary Phytochemical analysis showed the presence of carbohydrate, glycoside, alkaloid, protein, amino acid, saponin, tannin and flavonoid. One of the major Isoquinoline alkaloid is Berberine. From the present investigation an attempt has been made to standardize aqueous extract of Berberis vulgaris on the basis %age Berberine content by RP-HPLC.