The study of solution-phase interactions between small molecules and immobilized proteins is of intense interest, especially to the pharmaceutical industry. An optical sensing technique, dual polarization interferometry, has been employed for the detailed study of a model protein system, namely, d-biotin interactions with streptavidin immobilized on a solid surface. Changes in thickness and density of an immobilized streptavidin layer as a result of the binding of d-biotin have been directly measured in solution and in real time. The results obtained from this approach are in excellent agreement with X-ray crystallographic data for the structural changes expected in the streptavidin-D-biotin system. The mass changes measured on binding d-biotin also agree closely with anticipated binding capacity values. Determination of the density changes occurring in the protein adlayer provides a means for differentiation between specific and nonspecific interactions.