The medicinal plant industry is under increasing scrutiny due to wide variance in active ingredient (AI) concentration from values claimed on labels. Reasons for this disparity include environmental and genotypic variation which influence AI concentration. St. John’s wort (Hypericum perforatum) is a popular herbal remedy which also exhibits marked variance in AI concentration among products. This study evaluated concentration changes of three biologically active metabolites of H. perforatum after exposure to UV light while plants were still vegetative. Treatments were performed with 55-day-old plants grown under 400 μmol m−2 s−1 PAR for 16 h a day. Three UV light treatments were evaluated: a single dose, a daily dose and an increasing daily dose. Concentrations of hyperforin, pseudohypericin and hypericin were monitored for 7 days after each treatment. A daily dose and an increasing daily dose did not produce significantly greater increases in secondary metabolites compared to single dose treatments. These results suggest the small but significant transient metabolite concentration increases in H. perforatum can be induced by UV light exposure. Information from this study can be useful in optimizing total biomass and metabolite production in controlled environments.