Hypericin was isolated from Diahyperon tincture for use as a standard sample. The chromatographic purity of the hypericin was confirmed by high-performance liquid chromatography/mass spectrometry. Hypericin has the only chromatographic peak with a retention time of 23.10 min and gave a characteristic signal for the molecular ion [M – H+]– with m/z 504.05 in the mass spectrum. Electronic absorption spectra of Diahyperon tincture and the MeOH extract of Hyperici herba (Hypericum perforatum) showed characteristic hypericin bands at 550 and 590 nm and a band at 665 nm that was due to the presence of chlorophyll. It was shown that excitation of hypericin in Diahyperon tincture at λex = 470 nm gave an emission spectrum with characteristic hypericin bands at 593 and 640 nm while the emission spectrum of the MeOH extract of Hyperici herba showed the hypericin emission bands and a weak chlorophyll emission band at 670 nm. It was found that spectrophotometric and spectrofluorimetric determinations of hypericin in Diahyperon tincture and in Hyperici herba gave similar results that were consistent with the standardized values of the hypericin content in the studied samples.