There are widespread concerns regarding gastric diseases and discomfort, such as heartburn and reflux. The common medicinal solution prescribed is the use of proton pump inhibitors (PPIs). PPIs function by blocking the gastric HK-ATPase, inhibiting gastric acid secretion and leading to a significantly higher pH in the stomach throughout digestion. However, the importance of a gastric acidic environment for food digestion, erosion, and enzyme activity cannot be overlooked, and the impact of PPIs on food digestion and nutrient release remains relatively unexplored. To address this knowledge gap, our study aims to propose an in vitro semi-dynamic digestion protocol under PPI mediation and investigate the effect of PPIs on the digestion of a mixed meal consisting of bread, cheese, and tomato. The PPI digestion protocol was developed based on the INFOGEST semi-dynamic digestion protocol and the reported influence of PPIs on the acidity and rate of gastric secretions in humans. Gastric fluid and rabbit gastric extract (RGE) were added continuously, and five gastric emptying steps were conducted. Consistently with the in vivo literature, a final targeted gastric pH of 4.2 could be obtained by reducing by half the concentration of gastric acid and its secretion rate compared to healthy conditions. The emptied digesta was analysed for the digestion of carbohydrates, proteins, lipids, and minerals using ion chromatography, OPA, thin-layer chromatography, and ICP, respectively. Results revealed that under PPI conditions, less arabinose, peptides, and minerals (Ca, Mg, P) were released during digestion, while other compounds showed similar patterns with the healthy condition. The observed effects on nutrient digestion are likely due to the higher gastric pH, resulting in less acidic erosion and modified enzymatic activities. These results are consistent with some of the well-known adverse effects of PPIs such as infections, hypomagnesemia, fractures, etc. In conclusion, this study presents a detailed semi-dynamic digestion protocol under PPI medication, shedding light on the effects of PPIs on macro- and micro-nutrients during gastric digestion. This protocol can serve as a valuable resource for researchers studying food digestion, contributing to a better understanding of the association between PPI intake and clinical side effects, ultimately promoting better health.