Objective: To optimize the therapeutic dose and time window of picrosede Ⅱ in cerebral ischemic injury in rats by orthogonal test.Methods: The cerebral ischemia models were established by bilateral carotid artery middle cerebral artery occlusion.The successful models were randomly grouped according to orthogonal experimental design and treated by injecting picroside Ⅱ intraperitonenally at different ischemic time with different dose.The reactive glial proliferation was evaluated by GFAP immounohistochemical staining and the apoptotic cells counted by TUNEL detection kit.The expressions of neuroglial markers protein S 100β were determined by immunohistochemisty assay and Western blotting and the S100β mRNA by RT-PCR.Results:Picroside Ⅱ could significantly suppressed the reactive glial proliferation, the expression of S 100β and neuronal apoptosis.The optimized time window and therapeutic dose of picroside Ⅱ in cerebral ischemic injury were ischemia 1.5h with 10mg/kg body weight according to the expression of S100β, 2.0h with 10mg/kg body weight according to the apoptotic cells and the expression of S100β mRNA.Conclusions: Picroside Ⅱ might reduce ischemic brain injury by inhibiting the expression of S 100β.Combined evaluation from the principle of lowest therapeutic dose with longest time window, the optimized composition of the time window and therapeutic dose ofpicroside Ⅱ in cerebral ischemic injury were ischemia 1.5-2.0h with 10mg/kg body weight.