The principal components, isoflavonoids and astragalosides, in the extract of Radix Astragali were detected by a high-performance liquid chromatography couple to electrospray ionization ion trap multiple-stage tandem mass spectrometry (HPLC-ESI-IT-MS(n)) method. By comparing the retention time (t(R)) of HPLC, the ESI-MS(n) data and the structures of analyzed compounds with the data of reference compounds and in the literature, 17 isoflavonoids and 12 astragalosides have been identified or tentatively deduced. By virtue of the extracted ion chromatogram (EIC) mode, simultaneous determination of isoflavonoids and astragalosides could be achieved when the different components formed overlapped peaks. And this method has been utilized to analyze the constituents in extracts of Radix Astragali from Helong City and of different growth years. Then the antioxidant activity of different samples has been successfully investigated by HPLC-ESI-MS method in multiple selected ion monitoring (MIM) mode, applying the spin trapping technology, and the Ferric Reducing Antioxidant Power (FRAP) assay was applied to support the result. The correlations of the isoflavonoids and astragalosides components and the antioxidant activities of Radix Astragali were summarized. The present paper demonstrates that HPLC-ESI-MS(n) is a powerful method for the characterization of the principal components and evaluation of the antioxidant activity of Chinese medicinal herbs.