Recent reports have indicated that trienzyme treatment before folate determination is essential to obtain the proper folate content in foods. Trienzyme treatment is performed by using α-amylase and protease for folate extraction from carbohydrate and protein matrices, and folate conjugase for the hydrolysis of polyglutamyl folates. We evaluated the conditions of pH and incubation time for the treatment with α-amylase and protease. Four food items, including fresh beef, white bread, cow's milk, and fresh spinach, were selected for this investigation. We found that optimal pHs for α-amylase treatment of beef and cow's milk were 7.0 and 5.0, respectively, whereas those for white bread and spinach were not distinctive at pHs from 2.0 to 7.0. The optimal incubation time for a-amylase was 4 h for fresh beef and cow's milk, whereas no distinctive optimal incubation period was found for white bread and fresh spinach. Our data indicate that the conditions for enzyme treatments vary de-pending on food items. Trienzyme treatment resulted in an increase of more than 50% in the mean folate content over folate conjugase treatment alone. It is necessary to treat food samples with not only traditional folate conjugase, but also with a-amylase and protease before folate deter-mination to obtain the actual folate content.