This article reviews in vitro and in vivo models for studying gastrointestinal digestion, focusing on their benefits, limitations, and applications. The study examines static and dynamic in vitro models, including unicompartimental and multicompartimental systems that mimic gastric and intestinal phases. Key static models replicate pH, enzyme addition, and incubation times, with variations in enzyme sources (porcine, human, fungal), fluid composition, and digestion duration. Dynamic models, such as the Human Gastric Simulator (HGS) and TNO Intestinal Model (TIM-1), simulate physiological processes like gastric emptying, acidification, peristalsis, and enzyme secretion. A newly developed dynamic system, DIDGI®, was validated for infant digestion using piglets, showing comparable protein hydrolysis kinetics in vitro and in vivo. Colonic models, such as SHIME, simulate the large intestine and microbial fermentation. In vivo models include rats and pigs—commonly used due to physiological similarities to humans, especially pigs for nutritional studies. Human studies use direct methods (e.g., nasogastric intubation, scintigraphy) and indirect methods (plasma nutrient analysis). Ethical and technical constraints limit human studies. The article highlights the importance of selecting models based on research objectives, noting that in vitro models offer reproducibility and lower ethical costs, while in vivo models provide higher physiological relevance. The COST Infogest network has promoted standardisation of static digestion protocols to improve comparability across studies. The findings support the use of dynamic in vitro models as reliable alternatives before in vivo testing. SUBTITLE: Comprehensive review of in vitro and in vivo gastrointestinal digestion models, including static and dynamic systems, their applications, and physiological relevance