In vitro cultures of Hypericum heterophyllum Vent. was established by using MS and MS-B5 medium contained plant growth regulators such as kinetin, BAP, IAA, TDZ and picloram. Flower, leaf, stem and in vitro samples (callus and in vitro plantlets) of H. heterophyllum were analysed by LC-MS/MS for their chemical contents such as quinic acid, gallic acid, (+)-catechin, ferulic acid, vanillic acid, p-coumaric acid, caffeic acid, and quercetin; moreover their radical scavening activities conducted by DPPH and ABTS methods were evaluated. Among all the analysed samples, the in vitro plantlets shown the highest antioxidant activity (IC50, 220 mu g/mL for DPPH and 254 mu g/mL for ABTS), probably due to the presence of phenolic acids and flavonoids, specifically the higher total phenolic content (64.4 mg GAE/g extract) than other samples. The phytochemical variation among all samples was discussed through principal component analysis (PCA) and hierarchical cluster analysis (HCA). The in vitro plantlets might offer possibilities for the production of high-value secondary metabolites as pharmaceuticals and food preservatives. This study is the first report on analyses and comparison of secondary metabolites and antioxidant activities in different plant parts and in vitro samples of endemic H. heterophyllum.