Euphorbia helioscopia L. has been used as a herbal remedy for cancer in mainland China. Euphornin is one of the main bioactive constituents with the maximal content of Euphorbia helioscopia L. A reversed-phase high-performance liquid chromatography method with evaporative light scattering detection (ELSD) was developed for the analysis of euphornin for better quality control of E. helioscopia L. A good calibration curve in double logarithmic coordinator for euphornin was obtained. The validation study showed high recoveries (>97.0%) and low coefficient of variation (<3.0%). The use of the method on different euphornin extract samples confirmed its effectiveness. It was shown that ELSD was an effective detection method for the analysis of the non-volatile diterpenes from plants used in traditional Chinese medicine. The evaluation of the cytotoxicity of euphornin to mice lung adenocarcinoma cells (LA795) suggested that euphornin was one of the constituents of E. helioscopia L. responsible for the cytotoxicity against carcinoma cells.