For decades, the traditional food folate extraction method involved two steps including heat treatment, to release folate from its binding proteins, and folate conjugase treatment, to hydrolyze polyglutamyl folate to monoglutamyl folate. However, a trienzyme-extraction method of food folate was developed in the mid 1990s. This method involves the use of alpha-amylase, protease, and folate conjugase and allows for a more complete extraction of folate trapped in carbohydrate or protein matrices in food than the traditional method. In the last several years, this extraction method became widely used. However, the method is not uniform among various investigators, and it may be difficult for a new investigator to select the most suitable method in his or her laboratory. Therefore, in the review presented here, we summarize a variety of trienzyme-extraction procedures that were used by various researchers and offer a recommended procedure for food folate extraction. It is our hope that the wide use of an appropriate procedure of the trienzyme-extraction method, in combination with a reasonable detection method, help in establishing accurate and reliable food-folate tables and that this, in turn, makes it possible to accurately assess folate intake in the general population.