Withania somnifera L. Dunal (Solanaceae) is an important medicinal plant in the Indian system of medicine and is commonly known as Ashwagandha in India. A simple, rapid, sensitive, and reproducible high-performance thin-layer chromatography (HPTLC) method was developed to simultaneously quantify seven major bioactive metabolites in W. somnifera. A total of 08 germplasm of W. somnifera were collected from different locations in Central India for the quantification of withanolides (withanolide B, withaferin A, withanamide B, and withanoside V) and flavonoids (quercetin, apigenin, and kaempferol) by HPTLC analysis, which was accomplished on silica gel 60 F-254 by using toluene-ethyl acetate-formic acid (7:4.5:0.3, V/V) as the mobile phase. The in vitro antioxidant potential was also evaluated by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and deoxyribose assay methods. The highest amounts of apigenin, withanolide B, withaferin A, and withanamide B were found in WS-7 (Chitrakoot, Madhya Pradesh), in WS-6 (Satna, Madhya Pradesh), and in WS-3 (Lucknow in Uttar Pradesh) percentage dry weight, respectively. This type of study underscores the significance of identified bioactive compounds as reference tools for the accurate identification and confirmation of W. somnifera herbal products, ensuring their quality and efficacy.