The adsorption of bile salts to milk phospholipid and phospholipid-protein monolayers at the air-water interface was studied under simulated intestinal conditions using a Langmuir trough, epifluorescence microscopy, and atomic force microscopy. Surface pressure changes were affected by temperature, initial surface pressure, and bile composition. The rate of addition of bile salts and the initial surface pressure of the monolayers had an impact on the microstructure of the mixed monolayers. The presence of proteins in monolayers at different ratios did not affect the surface pressure change upon addition of bile. However, at 20 °C, the addition of bile to phospholipid and phospholipid-protein monolayers led to different features with branching and clustering of liquid-ordered domains and possible formation of bile salt-rich areas within liquid-ordered domains. This study provides a basic understanding of the interfacial changes occurring at the surface of milk fat globules and milk phospholipid liposomes during their passage in the duodenum.