The aim of this study was to identify and characterize groups of antioxidant active naturally from methanol extract of the stem of Anredera cordifolia through antioxidant assay-guided separation. The methanol extract (ME) displayed the highest antioxidant activity, compared to ethyl acetate extract (EE) and hexane extract (HE) using cyclic voltammetry method. Ascorbic acid was used as a positive control in this research. The repeated fractionation of active ME by MPLC and HPLC analyses yielded a constituent with strong antioxidant property. The active constituent (RNV-1) was predicted as a flavonoid type based on the UV spectrum data. The antioxidant activity results exhibited that RNV-1 had a coefficient of antioxidant activity (K) sligtly higher than K of ascorbic acid with the values of 0.055 and 0.052 respectively. It was found that RNV-1 demonstrated an excellent antioxidant activity in comparison with the standard antioxidant activity through antioxidant assays-guided separation. The results obtained suggested that the stem of A. cordifolia can be used potentially as a source of natural antioxidants by contributing beneficial health effects.