Inhibitions of α-amylase and α-glucosidases have been approached to attenuate the glucose-spike after food ingestion by regulating the glucose absorption. In this study, whole enzyme mixture (WEM) was applied to optimize the α-glucosidase inhibitory model by pharmaceutical inhibitors instead of crude enzyme solutions (CES) from rat intestinal acetone powder (RIAP) which is currently used in in vitro assay. WEM was reacted with various types of glycemic carbohydrates at 37°C with different concentrations of α-glucosidase inhibitors (acarbose, voglibose, and miglitol) in 100 mM phosphate buffer (pH 6.0). Based on the glucose generation rate, reaction times with α-glucosidase inhibitors were 1 and 6 h for maltose/maltodextrin and sucrose/isomaltulose, respectively. Also, the inhibition property during the hydrolysis of various types of substrates by WEM were clearly different compared to CES. The results support that there were different amounts and ratios of individual α-glucosidases between WEM and CES, and the use of entire α-glucosidases were required to determine the α-glucosidase inhibitory effects as well as the glycemic-carbohydrate digestion property more accurately. Thus, this designed-inhibition model with entire small intestinal α-glucosidases shows promise to determine the inhibitory effects more precisely compared to current assay with CES, and it can be applied to screen the natural/synthesized α-glucosidase inhibitors for controlling starch digestion rates with glucose delivery into the body in vitro level.