The aims of this study were quantitative determination of the bio-active compounds and to evaluate the in vitro antioxidant activities of methanolic extract of soy mixed wheat flour and traditional wheat flour. Soy mixed wheat flour was developed by combination of processed soy flour and wheat flour. Total phenolics, flavonoids, tannin, and proanthocyanidin contents of the crude methanolic extract were determined by ultraviolet-visible spectrophotometer. Further individual bio-active compounds were determined by high-performance liquid chromatography-diode array detector and showed (+)-catechin, vanillic acid, caffeic acid, (–)-epicatechin, p-coumaric acid, rutin hydrate, and ellagic acid in soy mixed wheat flour but only rutin in traditional wheat flour. The soy mixed product showed significantly higher concentration of bio-active compounds than traditional wheat flour. Antioxidative activities were measured through different in vitro models: phosphomolybdenum blue method, FeCl3 reducing power, ABTS scavenging activity, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity, N,N-dimethyl-1,4-diaminobenzene free radical scavenging activity, reducing power, metal chelating power and super oxide scavenging ability. All results of in vitro antioxidant models revealed that the soy product showed significantly higher antioxidant activities than traditional wheat flour. These results suggested that soy mixed wheat flour can play the greater roles than the traditional wheat flour for different physiological activities in human body due to the presence of greater amount of bio-active compounds and can be considered as a potential antioxidant containing flour for human consumption than the traditional wheat flour.