A silica gel column chromatography method is established for the isolation and purification of euparin and 12,13-dihydroxyeuparin from Radix Eupatorii Chinensis. For the first time, a high-performance liquid chromatography (HPLC) method is developed to determine simultaneously two benzofurans with UV absorptions at 240 nm. The analysis is performed on a Diamonsil C₁₈ column with a gradient solvent system of acetonitrile and aqueous phosphoric acid (0.2%, v/v) at a flow-rate of 1.0 mL min⁻¹. All calibration curves reveal good linearity (r² > 0.9998) within the tested concentration ranges. The relative standard deviations for intra-day and inter-day are less than 2.0% and the recoveries range from 98.32% to 103.68% with relative SDs less than 2.0%. This method is successfully applied to quantitate two benzofurans in Radix Eupatorii Chinensis. Therefore, the new HPLC method is proven to be reliable and suitable for the quality control of Radix Eupatorii Chinensis.