Berberine is an iso-quinoline alkaloid and is a major constituent of the species Berberis aquifolium (Berberidaceae.). Analytical thin layer chromatography was carried out on silica gel GF254TLC plates and the chromatoplate was developed with n-proponol: Formic acid: water (90:01:09v/v) as mobile phase. In this study isolation of berberine was achieved by preparative TLC and the compound thus isolated was characterised by Ultraviolet, Mass spectral analysis. Mass spectral data shows molecular ion peak of m/z=366. An isocratic RPHPLC method was developed for the estimation of berberine from mother tincture of Berberis aquifolium. The chromatographic separations was achieved by RP-C18 Column (250 mm X 4.6 mm, 5μ), Shimadzu LC-20AT Prominence liquid chromatograph and a mobile phase composed of 0.1%tri fluoro acetic acid:acetonitrile (60:40v/v). The flow rate was 1.0 ml/min and the analyses of column effluents were performed using UV-Visible detector at 344 nm. Retention time of berberine was found as 5.133 min. This method has obeyed linearity over the concentration range of 2-12μg/ml and the regression coefficient obtained from linearity plot for berberine was found as 0.997. RP-HPLC method was validated in pursuance of ICH guidelines.