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Considerable interest has developed in the role of flavonoid consumption in human health. In wheat, flavonoids are localized in the germ, particularly in the embryonic axis. The germ of wheat has a high nutrient density. In addition to phenolic compounds, germ is a valuable source of protein, fiber, and certain vitamins and minerals. With the growing interest in the role of flavonoids in disease prevention, it is important to gain a better understanding of the level and types of flavonoids consumed in our diets. Although wheat is not a particularly rich source of flavonoids, the high per capita consumption of flour in the typical American diet, makes the study of wheat flavonoids an important endeavor. We developed a reverse-phase HPLC procedure for the routine separation and quantitation of wheat flavonoids. The method was used to track the flavonoid distribution in six separate millings from the KSU pilot mill. Additionally, the development of a faster, more convenient method for quantitating flavonoid-containing tissue via digital fluorescence imaging (DFI) was investigated. The quantitative HPLC data was used as a basis for calibrating the DFI instrument. This research provides information about the quantity and types of flavonoids typically found in flour, as well further understanding of the distribution of germ in the milling process.