Agitated shoot cultures of three Hypericum perforatum cultivars: ‘Elixir’, ‘Helos’ and ‘Topas’ were established and maintained on Linsmaier and Skoog (LS) and Murashige and Skoog (MS) media containing varying concentrations (0.1–3.0 mg l−1) of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA). In methanolic extracts of the biomass, the amount s of free phenolic acids and cinnamic acid were determined by HPLC. Six of the seventeen compounds analyzed were detected in all the extracts: 3,4-dihydroxyphenylacetic acid, p-coumaric acid, protocatechuic acid, syringic acid, and two depsides—chlorogenic and neochlorogenic acids. The amounts of individual compounds and the total amount of phenolic acids depended on the media variants used and the increases ranged from 1.2- to 15.0-fold and from 1.2- to 1.9-fold, respectively. The maximum total amounts in the biomass of ‘Elixir’, ‘Helos’ and ‘Topas’ were 212, 222, and 191 mg 100 g−1 DW, respectively. The main metabolites in all the cultivars were neochlorogenic acid (max. 118.81 mg 100 g−1 DW) and 3,4-dihydroxyphenylacetic acid (max. 129.29 mg 100 g−1 DW). Three metabolites that also accumulated in high amounts were protocatechuic acid (max. 21.43 mg 100 g−1 DW), syringic acid (max. 23.63 mg 100 g−1 DW) and chlorogenic acid (max. 18.79 mg 100 g−1 DW). The richest s ource of the estimated compounds and hence a potential biotechnological platform were the shoots of ‘Helos’ cultivated on LS and MS media containing low concentrations of NAA and BA (0.1–1.0 mg l−1).