This study reports the first quantification study of pseudohypericin (PsHyp) and hypericin (Hyp) in Hypericum undulatum Schousb. ex Willd in vitro cultures developed by a Portuguese company. Both compounds were quantified by high-performance liquid chromatography and their levels were compared with those in commercial samples of Hypericum perforatum. PsHyp was found to be the major naphthodianthrone of H. undulatum, with an average ratio of 3.73:1 compared to Hyp. Significant statistical differences were found between the content of Hyp and PsHyp in H. undulatum regenerated shoots compared to in vivo samples. The mean concentration of total Hyps varied from 178.41 to 358.93 µg g(-1) dry extract in H. undulatum regenerated shoots, which is on average two to three times less than naphthodianthrone levels found in H. undulatum in vivo and H. perforatum commercial samples. However, none of the analysed samples presented the levels of Hyps required by the European and United States Pharmacopoeias.