Abstract: Recently antioxidant properties of plants have been found to be correlated with the defense of oxidative stress. In this respect flavonoids and other phenolic compounds have gained considerable attention. Calotropis gigantea has been widely used for its various pharmacological activities including cancer studies. In this study, the evaluation of antioxidant activity of methanolic extract of the flowers of C. gigantea has been done by in vitro method. The antioxidant activity of flower extract was evaluated by determining lipid peroxidation method, total phenolics contents was determined according to the Folin-Ciocalteu method and total flavonoid content by aluminium chloride colorimetric method. The results of the study showed that the methanolic extract of flowers of C. gigantea possesses antioxidant activity and it can be known by the protection of the cells. The total phenolic and flavonoid content was determined and expressed in term of gallic acid equivalent and quercetin equivalent, respectively. The results of the present study clearly showed that the flower extract of C. gigantea demonstrated antioxidant activities and it may act as potential antioxidant sources for human biological system.