The trienzyme digestion including protease, [alpha]-amylase, and conjugase for theextraction of total folate from cereals was optimized using response surfacemethodology. CRM121 wholemeal, oat, triticale, and buckwheat flours were used tosuggest an optimized digestion. Significant effects were determined for the incubationtime of [alpha]-amylase (p < 0.05) and conjugase (p < 0.01), while PronaseR digestion wasnot significant (p > 0.01) for extraction of total folate from the cereals. An optimumtrienzyme digestion time of PronaseR, [alpha]-amylase, and conjugase are 1 h, 2.5 h, and 6h, respectively. A differential assay of folic acid and total folate is used to determine [mu]gDFE in breakfast cereals (676 to 2184 [mu]gDFE/100g) and snack samples (15 to 394 [mu]gDFE/100g). To provide better information to customers, [mu]gDFE values should bedeclared on labels. Also, all assayed values of breakfast cereals exceeded labeldeclarations. Thus, exact labels on breakfast cereals will be needed.