Flavonoids with the quercetin structure are widely distributed throughout the plant kingdom. Some effects such as their anti-oxidative and radical scavenging capacities are broadly discussed in literature. Furthermore, some Hypericum flavonoids show activity in depression-relevant animal model assays. So far, only one study concerning the pharmacokinetic profile of Hypericum perforatum (St. John's wort, SJW) flavonoids has been reported, but no data concerning their bioavailability in the CNS is on-hand. Thus, we developed a method for the quantification of quercetin, tamarixetin and isorhamnetin, both metabolites of quercetin, in very low concentrations in the rat brain in order to investigate the ability of flavonoids to cross the blood-brain barrier. The brain samples for analysis were taken 4h after feeding an oral dose of an alcoholic SJW extract or pure isoquercitrin. We found the presence of quercetin and isorhamnetin/tamarixetin after feeding a SJW extract at 7 ng/g brain and 35 ng/g brain, respectively. In addition, we examined blood plasma taken from the same rats to correlate plasma and brain levels. The plasma levels were 350 ng/mL for quercetin and 1006 ng/mL for isorhamnetin/tamarixetin after intake of SJW extract.