Ethnopharmacological relevance: Hypericum perforatum (St.Johns wort) is an important antidepressant herb used in clinic and commonly prescribed for mild depression.Hyperoside is one of the major components of Hypericumperforatum and is also detected in many plant species such as Abelmoschus manihot, Black Currant, Rosa agrestis, Apocynum venetum and Nelumbo nucifera.Aim As hyperoside showed CNS (central nervous system) protective activity (e.g.antidepressant-like effect), the possibility of hyperoside or its metabolites to reach CNS should be investigated.Moreover, the pharmacokinetics profile of hyperoside or its metabolites in rat brain should be studied for further elucidating the mechanism of hyperoside action on CNS.Material and Methods A simple method for simultaneous determination of unbound hyperoside and its metabolite 3-O-methyl-hyperoside in rat brain was developed by using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) and microdialysis technique.This method was applied for pharmacokinetics study of hyperoside and 3-O-methyl-hyperoside in rat brain after intragastric (i.g.) and intraperitoneally (i.p.) administration of hyperoside in vivo.Results Results showed that neither hyperoside nor its metabolites were detectable in rat brain after i.g.administration but both compounds could be detected after i.p.administration.Considering the activity of hyperoside through both i.g.and i.p.administration, our results implies that the active components of hyperoside in vivo might be different.Therefore, further studies are needed to identify the active components of hyperoside in vivo through these two different routs.Moreover, non-oral administration route (e.g., i.p.) should be further investigated and be explored to obtain higher bioavailability and better activity for hyperoside.Our results also showed that the real existing form of hyperoside in rat brain were hyperoside and its methylated metabolite with maximum concentration to be 63.78 ng/mL and 24.66 ng/mL after 20 mg/kg i.p.administration, respectively.Therefore, a more reasonable concentration of hyperoside should be considered in in vitro assay to reflect the real situation of hyperoside concentration in vivo.Conclusion Due to the widely use of herbal remedies contain hyperoside, our investigation will contribute for further clarifying the action of this substance.Moreover, this method will be applied for clinic pharmacokinetics study of hyperoside and its metabolite as well as herbs that contain hyperoside.