In our study, we extracted alpha-glucosidase enzymes from human gut cells and measured the ability of these enzymes to break down sucrose, maltose or isomaltose in the presence of flavonoids and acarbose. A high-performance ion chromatography system was used to measure the sugars present, especially glucose, in the experiments. The inhibitory potentials of flavonoids were calculated based on the rate of glucose produced when measuring the activity of the alpha-glucosidase enzymes. Our approach using human intestinal cells and advanced and sensitive technology is superior to previous studies assessing the inhibitory potentials of flavonoids and provides more accurate outcomes. This study highlighted that flavonoids are inhibitors of human sucrase, maltase and, notably, isomaltase, providing a rational basis for using flavonoids from dietary sources as antidiabetic compounds.