The present study was conducted to determine the phenological changes in the content of main flavonoids, namely amentoflavone, hyperoside, isoquercitrin, quercitrin, quercetin, avicularin, rutin, (+)-catechin, and (-)-epicatechin in different tissues of Hypericum pruinatum, a promising medicinal herb of the Turkish flora. The wild-growing and greenhouse-grown plants were harvested at vegetative, floral budding, full flowering, fresh fruiting, and mature fruiting stages and dissected into stem, leaf, and reproductive tissues, which were dried separately and subsequently assayed for flavonoid contents by high performance liquid chromatography (HPLC). Chemical contents in whole plants increased during plant phenology and were similar quantitatively for both wild and greenhouse-grown plants. Depending on development stages, reproductive parts produced higher amounts of rutin, quercetin, (+)-catechin, (-)-epicatectin, and amentoflavone; however, leaves accumulated the highest level of hyperoside, isoquercitrin, quercitrin, and avicularin. The present results indicated a close relationship between flavonoid content in plant parts and phenological development of plants. It is suggested that the raw plant should be harvested during flowering for medicinal purposes.