The gut microbiota plays a key role in gastrointestinal immune and metabolic functions and is influenced by dietary composition. An in vitro protocol simulating the physiological conditions of the digestive system helps to study the effects of foods/biocompounds on gut microbiome and metabolome. The Dynamic-Colonic Gastrointestinal Digester consists of five interconnected compartments, double jacket vessels that simulate the physiological conditions of the stomach, the small intestine and the three colonic sections, which are the ascending colon, transverse colon and descending colon. Human faeces are required to reproduce the conditions and culture medium of the human colon, allowing the growth of the intestinal microbiota. After a stabilization period of 12 days, a food/biocompound can be introduced to study its modulatory effects during the next 14 days (treatment period). At the end of the stabilization and treatment period, samples taken from the colon compartments are analysed. The 16S rRNA gene analysis reveals the microbiota composition. The untargeted metabolomics analysis gives more than 10,000 features (metabolites/compounds). The present protocol allows in vitro testing of the modulatory effects of foods or biocompounds on gut microbiota composition and metabolic activity.