An effective method for simultaneous separation of four flavonoids and two astragalosides was developed by semi-preparative high-performance liquid chromatography in this work. A crude sample was firstly cleaned-up from ethanol aqueous extract of Radix Astragali by LS-300B resin-based column chromatography and was further isolated by semi-preparative LC with a gradient elution of water-methanol. Six compounds including calycosin (3.5 mg), formononetin (2.6 mg), (6aR,11aR)-10-hydroxy-3,9-dimethoxypterocarpan (2.6 mg), (3R)-7,2′-dihydroxy-3′,4′...