Friction force spectroscopy (FFS) has been applied to study the tribological properties of β- and κ-casein layers on hydrophobic substrates in aqueous solutions. Nanometer-sized imaging tips were employed. This allowed exerting and determining the high pressures needed to remove the layers, and registering the topographic evolution during this process. Both β- and κ-casein layers showed similar and not particularly high initial frictional responses (friction coefficient of ~1 when measure with a silicon nitride tip). The pressures needed to remove the layers were of the same order of magnitude for both proteins, ~1e8 Pa, but slightly higher for those composed of β-casein. The technique has also shown to be useful in studying the two-dimensional lateral diffusion of the proteins and the wear on the layers they form.