We evaluated the effect of protease, α-amylase, and folate conjugase treatments on apparent folate content in four food composites designed to be low in folate (31.2 μg/day calculated according to previously published food folate tables) for the determination of folate requirements in humans. Protease and α-amylase treatments were performed for 16 and 4 h, respectively, at pH 4.1 and chicken pancreas folate conjugase (FC) for 3 h at 37 °C. Folate was determined by microbiological assay using the Lactobacillus casei method. Mean folate content in these menus was 56 μg/day after treatment with FC alone. However, means were 90 μg/day with protease and FC, 126 μg/day with α-amylase and FC, and 152 μg/day with α-amylase, protease, and FC treatments. Tri-enzyme treatment appears to be essential to determine food folate content accurately. Food folate tables should be revised using these enzyme treatments to accurately establish the dietary folate requirements in humans.