For a better understanding of biofilm establishment, adhesion forces of proteins on immobilized protein layers were investigated by scanning force spectroscopy (SFS). For this purpose the experimental setup to immobilize protein layers on the surface is important. The influence of the method chosen to immobilize the proteins on the surface was examined and afterwards the effect of variations in pH and contact time on the measured adhesion forces in protein–protein interaction was analyzed. The adsorption of bovine serum albumin (BSA) on BSA-coated silicon was used here as a model system. To coat the substrate, free adsorption, crosslinking with glutaraldehyde, and covalent protein binding to the surface after silanization were used. For the latter different deposition methods of the silane were compared. Depending on the immobilization method the measured protein–protein interactions show distinct differences. Covalent attachment of proteins is a promising approach to attain stable, pre-adsorbed protein layers for SFS measurements. The adhesion forces of a BSA-coated cantilever on such layers seem to be dominated by electrostatic interactions of the molecules.