Atomic force microscopy (AFM) is a widely used technique for imaging sample topography and measuring intermolecular forces between a probe and a surface. The evaluation and quantification of molecular interactions of colloidal biomolecules using this technique is of critical importance to the food industry, as the interfacial forces of colloids determine the texture, viscosity, and stability of foods. To measure these interactions, functionalized substrates and cantilever probes are necessary, and a robust protocol is essential for ensuring reproducible measurements. In this chapter, we present a protocol for the AFM technique to obtain information on intermolecular forces, including the distance and energy required for interactions to occur. We also discuss theoretical models, such as the Derjaguin–Landau–Verwey–Overbeek (DLVO) approach, which describes molecular interactions and the contribution of different types of molecular forces. The combination of the AFM technique with the study of the interface in food systems enables the manipulation and improvement of products to enhance food safety, increase shelf life, and improve food stability.