Protein-lipid interactions play an important role in a variety of fields, for example in pharmaceutical research, biosensing, or food science. However, the underlying fundamental processes that govern the interplay of lipids and proteins are often very complex and are therefore studied using model systems. Here, Langmuir monolayers were used to probe the interaction of a model protein with lipid films at the air-water interface. The protein β-lactoglobulin (βlg) is the major component in bovine milk serum, where it coexists with the milk fat globular membrane. During homogenization of milk, βlg adsorbs to the interface of lipid fat globules and stabilizes the oil-in-water emulsion. pH and ionic strength of the subphase had a significant effect on the surface activity of the protein. Additionally, by using lipids with different charges, it could be shown that the interactions between βlg and a phospholipid layer were driven by hydrophobic as well as by electrostatic interactions. βlg preferentially interacted with phospholipids in an unfolded state. This could be either achieved by denaturation at the air-water interface or due to electrostatic interactions that weaken the intramolecular forces of the protein.