Methodology for studies of effects of heating multilayers of human serum albumin (HSA) and anti-HSA is presented. Multilayers of anti-HSA were prepared on silicon substrates and studied with infrared spectroscopic ellipsometry equipped with a heat stage. The refractive index N = n + ik and the layer thickness are determined and the amide bands are analyzed. It is found that HSA/anti-HSA multilayers are stable for shorter times at temperatures above 100 °C, except for small thickness changes. Also pilot studies of effects of heating monolayers of proteins adsorbed on gold substrates is presented.