Gentiopicroside, mangiferin, sweroside, and isoorientin, the bioactive constituents of Swertia mussotii Franch, have various pharmacological effects, and are used in particular for treating liver disorders. However, efficient methods for their separation are not currently available. In this study, these bioactive compounds were detected in S. mussotii extracts using high-performance liquid chromatography coupled with mass spectrometry, and separated using a combination of high-speed counter-current chromatography and Sephadex LH-20 column chromatography. Their structures were determined by using H-1 and C-13 nuclear magnetic resonance spectroscopies. The results show that this method is effective for the separation and purification of bioactive compounds from S. mussotii.