In the Langmuir monolayer technique, a single layer of molecules is formed on a water subphase. This approach was used to mimic the antitumoricidal lipid-protein complex of oleic acid and bovine α-lactalbumin called the BAMLET complex. Our previous studies have shown that at the interface, the BAMLET complex is stabilized by the hydrophobic forces supported by the hydrogen bonds. This study provides an insight into the influence of calcium ions and the experimental conditions (temperature and subphase pH) on the stability of the complex at the interface. The Langmuir technique was expanded using a dosing pump to exchange the subphase and deliver additional substances to the system. We investigated the interactions between oleic acid monolayer and α-lactalbumin in the presence of Ca2+ in the bulk and the effect of varied experimental conditions on the complex stability. The role of calcium ions in this system is important because (in addition to low pH and relatively high temperature) it affects the conformational changes within the protein molecule and facilitates the transition of α-lactalbumin into the molten globule state. A partially unfolded state is required to form the BAMLET complex. We found that the mixed monolayer spread at the interface is stable despite drastic changes in the process conditions and remains stable even after the subphase exchange. This study of molecular interactions explored by the Langmuir technique with peristaltic pump enabled to understand the role of Ca2+ in BAMLET complex formation.