Radix astragali (Huangqi in Chinese) is a well-known traditional Chinese medicinal herb that has been used clinically in China for centuries to cure various diseases. To profile the antiviral constituents of this herb, a high-performance liquid chromatography-diode array detector-electrospray ionization-tandem mass spectrometry (HPLC-DAD-ESI-MS/MS) analytical method was developed to separate and determinate the active part of the extract of Radix astragali, which showed potent inhibition of several viruses. By comparing their retention time and MS data with those obtained from the authentic compounds and the published data, a total of 18 compounds, comprising 11 flavonoids and 7 saponins, were identified. This study provides an approach to rapidly characterize bioactive constituents in traditional Chinese medicines (TCMs).