Grifola frondosa, commonly known as 'Maitake', has been used traditionally by Chinese and Japanese in their curative herbal medication. The present study was undertaken to provide quality control standardization based on microscopic, physico-chemical and chromatographic features and to evaluate antioxidant activity. The physicochemical parameters such as loss on drying, ash value, fluorescence analysis and methanol soluble extractive were determined by standard methods. HPTLC profile of methanolic extract was carried out by scanning along the range of 200 to 400 nm. The Rf values were noted as an important parameter for identification of the mushroom. HPLC profile of the extract detected presence of 38 peaks. Preliminary mycochemical analysis indicated presence of cardiac glycoside, flavonoid, phenol, saponin and terpenoids. Quantitatively the major bioactive components were present in following order of phenol> flavonoid> ascorbic acid> β-carotene> lycopene in methanolic extract. Along with, DPPH radical scavenging activity (EC50 value 666 μg) and total antioxidant capacity (18.64 μg AAE/mg of extract) were determined. The distinctive features established in this work are steps towards identification, standardization and quality control of the medicinal mushroom.