Dietary fat has long been recognized as an essential component in nutrition. However, most of the lipids present in food need to be converted into more bioavailable compounds. Lipases have a crucial role in converting triglycerides into more polar lipids with increased water solubility and a tendency to form micelles. However, the surface active molecules generated by lipolysis may have a detrimental effect on the interfacial biocatalysis. In the present work we evaluate the interfacial properties of lipase-generated molecules during fat digestion. By using the pendant drop technique we assessed the amphiphilic character of fatty acid salts, monoglycerides, and diglycerides as individual surfactants and mixtures. The experimental results are fitted with a mathematical model, which assists in the determination of the interfacial properties of the surfactants. Our results show that monoglycerides have considerably higher interfacial activity than fatty acid salts and diglycerides. Therefore, the interface will soon be dominated by monoglycerides. The pH dependency of the interfacial activity of fatty acids is also explored in the current work. We believe that our results can contribute to a better understanding of the complex interfacial phenomena occurring during fat digestion.