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Various chromatographic techniques have been designed to estimate soy isoflavones in finished products as well from Glycine max(L.) Merrill. As an attempt to develop a simple, accurate, and validated method for routine analysis of daidzein and genistein, the major isoflavones as an established standardization technique, a high-performance thin-layer chromatographic (HPTLC) method has been developed. A comparative analysis of individual isoflavone (daidzein and genistein) contents in different varieties of G. maxCO-2, CO (soy) 3, and local white was also carried out. Extraction efficiency of the targeted isoflavone from the seed matrix with organic solvents using ultrasonication technique was studied. The methanolic extract of the soybean seeds was taken for method development and validation procedures by HPTLC in which separation was achieved on silica gel 60 F254HPTLC plates using chloroform—methanol (20:1 v/v) as the mobile phase. The individual isoflavones and protein contents were estimated in three different varieties of soybean in which CO-2 variety has shown the highest content.