To detect and identify natural antioxidants in Swertia chirayita with protective effect against cerebral infarction, a screening method, using column chromatography and cerebral ischemia-reperfusion injury in rat, was developed. Seventeen compounds were purposefully separated and identified by Nuclear Magnetic Resonance, Fourier Transform Infrared Spectroscopy, Ultraviolet Spectrum, and Mass Spectrometry. The purified compounds were further screened by radical scavenging activity and cerebral ischemia-reperfusion injury in rats. Two compounds showed apparent radical scavenging activity and neuroprotective activity. The two compounds were identified as 1-hydroxy-2,3,4,6-tetramethoxyxanthone and 1,5,8-trihydroxy-3-methoxy xanthone, and were preliminarily considered as primary natural neuroprotective antioxidants in Swertia chirayita. These two compounds (20 mg kg−1) markedly decreased infarct size to below 5%, and also caused a significant improvement of activities of superoxide dismutase (SOD) (92.90 ± 11.19 U ml−1), glutathione peroxidase (GSH-Px) (122.58 ± 12.31 μmol mg−1) and a decrease in the content of malondialdehyde (MDA) (3.98 ± 2.00 nmol ml−1) in serum. The two compounds showed strong capability for protective effects against cerebral damages induced by ischemia-reperfusion, and the protective effect may be related to the inhibition of lipid peroxidation. The use of the screening method based on tracing separation and ischemia reperfusion would provide a new way for detection of radical-scavenging and natural neuroprotective compounds from Swertia chirayita or complex matrices.