Silybin is a characterising compound present in the plant Silybum marianum, used as an hepatoprotective. A simple, accurate and sensitive HPLC method was developed for estimation of silybin. The chromatographic separation was carried out on RP C18 (Waters) column (250 × 4.6 mm, 10 μm) using mobile phase, phosphate buffer: water (65:35 % v/v) pumped at a flow rate of 1 ml/min. The eluent was monitored at 288 nm. The method is specific and linear over the range of 10-200 ng/ml. The method was statistically validated for precision, accuracy, robustness and recovery. The HPLC method can be applied for identification and on of silybin in herbal extracts of Silybum marianum.