The well-known relation between food and bioactives explores the great prospective of innovative and novel food to maintain or improve health, increasing the demand investigating advance products with medicinal effect. Withania somnifera also known as ‘Ashwagandha’ in India, is one of the most utilized plants in different ancient medicine systems. The present work is an effort to develop a knowledge-based concept and a quality control approach for the development of functional foods. The study took into account the standardization criteria and examined how different geographies or different field sites (of India) would influence the phytoconstituents and their distribution in the different samples of W. somnifera. Phytoconstituents level were strongly influenced by soil quality, cultivar, and growing season. An increase in phenolic content, flavonoid content, antioxidants and the quality yield, were all correlated with increased phytoconstituent concentrations. The highest hydro-methanolic extract yield (by reflux (Soxhlet), sonication (20 min) and maceration (16 h) was from the sample collected from Rajasthan (18.61%, m/m) followed by West Bengal (11.21%) and Madhya Pradesh (12.67%) respectively. The methanolic extract of Rajasthan sample (produced by the reflux (Soxhlet) extraction method) had the highest concentrations of flavonoids (65.32 mg quercetin equivalent (QE)/g) and the total phenols (60.35 mg gallic acid equivalent (GAE)/g), while Madhya Pradesh sample (distilled water reflux (Soxhlet)) extract had the highest antioxidant content (74.13 %). High performance thin layer chromatography (HPTLC) method was also utilized for identification of withanolides A. Chromatographic separations were carried out using V(ethyl acetate):V(toluene): V(acetic acid) = 9.1:1:0.6 as the mobile phase. The gas chromatograph-mass spectrometer (GC-MS) study of hydro-methanolic Soxhlet extract of W. somnifera from 3 different regions of India was done and the total phytochemical substances identified were 132 in sample from Rajasthan, 107 in sample from West Bengal, and 164 in sample from Madhya Pradesh respectively. To conclude, the present study demonstrates how, within a framework of integrated agricultural practices, yield and phytochemicals merge into a new paradigm of functional foods.