This study evaluated the anti-oxidant activity of avicularin and isovitexin from Lespedeza cuneata using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl (OH) scavenging assays. The results showed that among the four fractions, the highest OH radical scavenging activity was exhibited by the n-butanol fraction. The DPPH radical scavenging activities of avicularin and isovitexin were higher than 50% at a concentration of 100 μg/mL. Moreover, one hundred micrograms per liter of avicularin and isovitexin exhibited effective anti-oxidant activity, with the OH radical scavenging rates being 87.54 and 91.48%, respectively. These results suggest that Lespedeza cuneata would be useful anti-oxidant agents from natural sources.