Oxidative stress related disorders occur as a result of free radicals in the cellular organs. Antioxidants provide protection contrary to oxidative stress by counteracting free radicals through the help of different bioactive constituents present in the plants. The aim of this study is to find out the quantitative estimation of phytochemicals (phenolics, tannins, flavonoid, non-tannins, alkaloid and saponins), in vitro antioxidant activity (2, 2-diphenyl-1-picryl-hydrazyl-hydrate, metal chelating, ferric reducing, superoxide dismutase and total antioxidant activity) of different parts of Cyperus rotundus. Along with this, the extraction of polyphenolic compounds by conventional and non-conventional methods and their optimization by response surface methodology technique were also studied. As the outcomes of the study, maximum amount of total phenolics content was found in the ethanolic fraction of stem (37.88±0.94 mg gallic acid equivalent/g). The root has shown the highest flavonoid (0.24±0.03 mg QAE/g) and non-tannin content (21.55±2.43 mg gallic acid equivalent/g) in the extract of petroleum ether. The total alkaloids and tannin content in flower and root i.e., 5.05±0.08 mg caffeine equivalent/g and 76.28±0.62 mg tannic acid equivalent/g was found to be better in aqueous extract respectively. The fraction of ethyl acetate has reported the greatest saponin content (39.2±3.8 mg gallic acid equivalent/g). In case of flower part half-maximal inhibitory concentration value of 2, 2-diphenyl-1-picryl-hydrazyl-hydrate and superoxide dismutase antioxidant activity varies from 0.57-1.90 mg/ml and from 0.053-1.401 mg/ml respectively. Antioxidant activity of each part of the plant was reported in the fraction of ethanol and water by fluorescence recovery after photobleaching (0.403±0.009-0.886±0.02 mg/ml), metal chelation activity (0.010±0.0-0.306±0.001 mg/ml) and total antioxidant capacity (0.030±0.0-0.317±0.002 mg/ml). Microwave assisted extraction method was found to be the suitable one for optimization of total phenolic content. So, the relationship between different parameter was determined through response surface methodology technique and evaluated by plotting the graphs. Polar extracts have found to show the maximum quantity of secondary bioactive metabolites and antioxidant activity as compared to non-polar one. And among the comparison between conventional and non-conventional methods, ultra sonication (nonconventional method) has shown the highest yield of bioactive phenolic compound. So, this study shows that Cyperus rotundus has wide range of secondary metabolites showing antioxidant properties that can be applied for different therapeutic applications.