St. John's Wort (Hypericum perforatum L.) was extracted with supercritical carbon dioxide (SC CO 2 ) under 10, 15 and 20MPa and at 313 and 323K. The influence of SC CO 2 density (400–800kg/m 3 ) was examined with respect to extraction yield as well as to hyperforin and adhyperforin content, which was determined using a highly selective LC/MS/MS analytical method. A high concentration (up to 526mg/g total extract) of two phloroglucinol derivatives was determined in this study showing that the absolute extracted amount of hyperforin and adhyperforin (mg per 1g of dry plant) is almost constant not depending on total yield of extract, i.e. on density of SC CO 2 . Determination of the rate of hyperforin and adhyperforin extraction at 313K and 10MPa showed that the maximal rates correspond to consumption of 7–8.5g of CO 2 per 1g of dry St. John's Wort.Investigation of ultrasonic extraction of St. John's Wort followed after pre-treatment of plant material (323K; 10MPa; 2h; without CO 2 flow) indicated that obtained extract contains a higher amount (mg/g plant material) of two most important pharmacological active compounds of St. John's Wort, hyperforin and hypericin, than the extract obtained by ultrasonic extraction only.