The research investigated the dynamics of released glucose from whole wheat flour during in vitro gastric and intestinal digestion. Two experiments were conducted. Experiment 1 (gastric digestion) was an enzymatic hydrolysis with a pepsin solution at pH 2.0 and 37°C for 120 minutes. In experiment 2, first step was followed by the hydrolysis with the multi-enzyme pancreatin (intestinal digestion), at pH 6.8 and 37°C for 4 h. The released glucose (RG) and the refractive index (RI) were determined from the supernatant obtained after centrifugation. Experiments showed an initial increase in the glucose released from 1 g wheat up to 0.472 mg/mL after 1 h of gastric digestion, then followed by decrease. The lowest value was recorded after 2 hours of gastric digestion (0.332 mg/mL). The concentration of the reducing sugars increased throughout the intestinal digestion from 0.549 mg/mL up to 1.990 mg/mL, as a result of starch hydrolysis to dextrins, maltose and then to glucose. The high positive correlation between RG and RI suggests that RI may be used as a simple and rapid method for estimating the released glucose during in vitro digestion.