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Abstract This is a study of microstructure changes and Soybean milk colloidal particles degradation during in vitro digestion. Soybean milk passed through the digestion stages of the Infogest protocol, while its colloidal particles were examined in terms of size decrease/free surface evolution kinetics and of surface charge. Colloidal protein break-down was determined in relation to the digestion stage, and the shape and structure of the soybean milk particles were monitored with confocal microscopy. Gastric digestion significantly influenced the soybean milk particles, increasing the particle size and decreasing absolute surface charge, hydrolyzing protein and accumulating aminoacids. Similar breakdown was not observed in the absence of pepsin, so the protein break-down can be primarily attributed to pepsin-induced hydrolysis. Correlation of the aminoacids release kinetics with the changes in surface area of the soybean milk proteins showed a direct relation between aminoacids release and solid-water interface formation. A sequence of events can be proposed where proteins are exposed and hydrolyzed throughout the simulated gastrointestinal tract as new interface (substrate) is created in the soybean milk colloidal systems. Highlights • The oral–gastric–intestinal digestion of soybean milk was studied in vitro. • The physical properties of the milk particles were affected by the enzyme presence. • Hydrolysis reduced inter-particle repulsions, causing irreversible aggregation of oil droplets. • Soybean milk were physically changed in absence of enzymes during in vitro digestion. [ABSTRACT FROM AUTHOR]