High-performance centrifugal partition chromatography (HPCPC) combined with ultraviolet detection was employed for the separation and purification of flavonoids from Glycyrrhiza. At the detection wavelength of 276 nm, two flavonoids, liquiritigenin-4'-apiosyl-glucoside and liquiritin, were successfully separated by HPCPC with an optimized two-phase solvent system composed of ethyl acetate-ethanol-water (1:0.1:1, v/v/v). The purity of these compounds was 95.0 and 97.1%, respectively, as determined by high-performance liquid chromatography. Their structures were identified by electrospray ion source mass spectroscopy (ESI-MS(n)) in the negative ion mode.