Background: Ophiopogonis Radix is a famous traditional Chinese medicine. It is necessary to establish a suitable quality control methods of Ophiopogonis Radix. Objective: To investigate the quality control methods of Ophiopogonis Radix by high-performance liquid chromatography (HPLC) coupled with evaporative light scattering detector (ELSD). Materials and Methods: A rapid and simple method, HPLC coupled with ELSD, was applied to determinate ruscogenin in 35 batches of Ophiopogenis Radix samples. Orthogonal tests and single factor explorations were used to optimize the extraction condition of ruscogenin. The content of ruscogenin in different origin was further analyzed by hierarchical clustering analysis (HCA). Results: The ruscogenin was successfully determined by HPLC-ELSD with a two-phase solvent system composed of methanol-water (88:12) at a flow rate 1.0 ml/min, column temperature maintained at 25°C, detector draft tube temperature at 42.2°C, nebulizer gas flow rate at 1.4 L/min, and the gain at 8. The result showed the good linearity of ruscogenin in the range of 40.20-804.00 μg/ml (R2 = 0.9996). Average of recovery was 101.3% (relative standard deviation = 1.59%). A significant difference of ruscogenin content was shown among 35 batches of Ophiopogenis Radix from different origin, varied from 0.0035% to 0.0240%. HCA based on the content of ruscogenin indicated that Ophiopogonis Radix in different origin was mainly divided into two clusters. Conclusion: This simple, rapid, low-cost, and reliable HPLC-ELSD method could be suitable for measurement of ruscogenin content rations and quality control of Ophiopogonis Radix.