The neuroprotective effect of Withania somnifera L. Dunal fruit extract, in rodent models, is known. Withanamides, the primary active constituents in W.somnifera fruit extract exhibited neuroprotective effects against β-amyloid-induced cytotoxicity in neuronal cell culture studies. Therefore, we investigated the blood-brain barrier permeability of withanamides in W.somnifera fruit extract in mice using HPLC coupled with high resolution quadrupole time of flight mass spectrometer (Q-TOF/MS) detection. Mice were administered with 250 mg/kg of W.somnifera extract by intraperitoneal injection, and the blood and brain samples analyzed by Q-TOF/MS detection. Four major withanamides were detected in brain and blood of mice administered with W.somnifera extract. The results suggested that the withanamides crossed the blood-brain barrier. These results may help to develop W.somnifera fruit extract as a preventive or therapeutic botanical drug for stress-induced neurological disorders.