Rat intestinal mucosal α-glucosidases could be a mammalian enzyme source to be used in in vitro carbohydrate digestion kinetic studies, and to further discern effects of substrate and inhibitor effects on the individual four enzymes – maltase, glucoamylase, sucrase, isomaltase. The commercialized rat intestinal powder enzyme comes with α-amylase contamination, and therefore needs to be purified for in vitro starch digestion. The objective of this study was to separate α-glucosidase from the crude rat intestinal powder by size-exclusion chromatography using a media with fractionation in the range of Mw 1 × 103 to Mw 1 × 105. Elution times of the α-glucosidases and α-amylase was found from 26.5 to 43.0 min ( expected molecular weight, 48-90 kDa, excepting a minor peak at 125 kDa) and from 43.0 to 63.0 min (expected molecular weight, 51–54 kDa), respectively. Good separation was achieved with specific enzyme activities of α-glucosidase (fraction range from 26.5 min to 43.0 min) of 44.7 (unit/mg protein) and negligible α-amylase activity (1.0 unit/mg protein). The results demonstrated that purified α-glucosidases from rat intestinal power with high α -glucosidase activity can be efficiently achieved for in vitro carbohydrate hydrolysis test.