An RP-HPLC method with photodiode array detection was established for the determination of major constituent, hyperforin in St. John’s Wort extract samples. The hyperforin was separated by using gradient mode consisting of 0.2 % formic acid in water and 0.2% formic acid in methanol at a flow rate of 1.0 mL/min. Under these conditions, a plot of integrated peak area versus concentration of hyperforin was found to be linear over the range of 1.0 – 100.0 μg/mL, with a relative standard deviation of 0.16 – 0.88%. The limit of detection was 10 ng on column and the limit of quantitation was 20 ng on column. The determination of the hyperforin content in a commercially available St. John’s Wort extracts exhibited a mean content of 2.0 – 27.0 % w / w. Recovery experiments led to a mean recovery rate of 98.12 ± 0.94 %. The proposed method is not time-consuming, sensitive and reproducible and is therefore suitable for routine analysis of hyperforin in herbal medicinal products.