We investigated the effect of Euphorbia supina extract on the production of cellular reactive oxygen species (ROS) in order to evaluate its antioxidative activity. The H₂O₂-induced ROS generation was measured with dichlorofluorescein-diacetate (DCFH-DA) assay. The acetone+methylene chloride (A+M) and methanol (MeOH) extracts dose-dependently decreased ROS production induced by H₂O₂ compared to the extract-free control during the 120 min assay. The MeOH extract showed a higher inhibitory effect on the A+M at all concentrations tested. All tested fractions showed a decrease in ROS production with increase in concentration. Treatments with n-hexane and 85% aqueous methanol (MeOH) fractions (0.5 mg/mL concentrations) showed 82% and 97% inhibition rate, respectively, suggesting that the 85% aqueous MeOH fraction had ROS scavenging effects. These results indicate that the 85% aqueous MeOH fraction inhibited cellular oxidation and may contain valuable active compounds.