Hypericum perforatum is a high value medicinal herb possessing a broad range of pharmacological activities. These medicinal properties are attributed to the presence of major compound hypericin. In current study, the effect of different growth hormones and media combinations along with various growth parameters including light and temperature were analyzed for hypericin production in in vitro grown shoot cultures of H. perforatum. Rapid method for micropropagation with an average of 36.3 shoots per explant in 5-6 days was developed. Highest hypericin content of 0.119 µg/mg was detected in shoots grown on MS medium containing sucrose (3%), IBA (3mg/l), KN (1mg/l) and agar (0.9%) at 25±2 °C under light conditions after two months of incubation. In addition, sustention of in vitro grown shoots after hardening under greenhouse conditions was also observed showing comparable hypericin content in hardened and field grown plants after 2 years. Therefore, current study reports the rapid method for in vitro multiplication for hypericin rich shoots of H. perforatum under optimized light and temperature conditions. To the best of our knowledge, this is the first report on comparative analysis of hypericin production in hardened and field grown shoots of H. perforatum. Hence, the outcome is an endeavor to meet the increasing industrial demands by providing quality rich raw material using tissue culture techniques.