Total folate content was determined by microbiological assay using Lactobacillus casei spp. rhamnosis (ATCC 7469) with a 96-well microplate technique. Using roasted peanut and red kidney beans as representative legume samples, response surface methodology (RSM) was supplied to optimize the trienzyme procedures for the determination of folate in legumes. After response surface regression (RSREG), the second-order polynomial equation was fitted to the experimental data. Ridge analysis showed that the optimal digestion times were <2 hr for $Pronase^{(R)}$ and $alpha$-amylase, and <5 hr for conjugase to obtain maximal folate values for legume samples. This study confirms that established digestion times for cereal products (AOAC Method 2004.05) of 3 for protease and 2 hr for $alpha$-amylase are applicable to legumes. Conjugase treatment can be reduced to 5 from 16 hr and the conjugase level to 5 from 20 mg per sample, providing significant cost saving.