Porous silicon layers with a one-dimensional lateral gradient in pore size are prepared by electrochemical etching and characterized by spectroscopic ellipsometry in the visible to near-infrared region. The ellipsometer is equipped with a micro-spot option giving a lateral resolution of approximately 100 μm. By matching multiple-layer-model calculations to the laterally-resolved variable angle of incidence spectroscopic ellipsometry data, the thickness variation along the gradient as well as the in-depth porosity profile is mapped. Upon exposure to a protein solution, protein adsorption occurs on top of the porous silicon layer. At the high-porosity region of the gradient also penetration of protein molecules into the porous layer takes place. Ellipsometry data are recorded after protein exposure and variations of protein adsorption along the porous silicon gradient is modeled as well as the in-depth profile of protein penetration.