Since 1985, AOAC Method 985.29 has been globally adopted as a standard method for determination of total dietary fiber in foods. Nevertheless, an aspect of AOAC Method 985.29 that needs to be improved is the laborious process to treat 3 enzymes separately at their individual proper pH, which is quite time-consuming. Several examinations have been carried out to resolve this problem. The characteristics of newly developed thermostable alpha-amylase, neutral protease, and amyloglucosidase were evaluated based on the pH-activity profile and the property of starch hydrolysis in comparison with those of the conventional enzyme reagents. These 3 developed enzymes were found to work under the same pH condition and to accomplish sufficient digestion for the typical 3 starches: soluble starch, corn starch, and wheat starch. The experimental results revealed that the dietary fiber determination in foods could be performed without pH adjustment in the enzymatic digestion process. The modified method will be greatly helpful in determining the total dietary fiber contents in food materials with less laborious work and with an accuracy equivalent to that of AOAC Method 985.29.