Human serum albumin (HSA) adsorbed to thin films of phase-segregated polystyrene (PS)-poly(methyl methacrylate) (PMMA) was examined under hydrated and dry environments with scanning transmission X-ray microscopy (STXM). Quantitative mapping of the protein and polymer components at 30 nm spatial resolution was achieved using near-edge X-ray absorption fine structure (NEXAFS) spectral contrast at the C Is edge. Under fully hydrated conditions (0.005 mg/mL HSA), adsorbed HSA thicknesses in excess of its crystallographic dimensions suggest bilayer adsorption to the polar PMMA regions. Upon washing, these loosely bound protein molecules adsorbed to PMMA were removed. Upon drying, the thickness of HSA on the nonpolar PS region decreased by similar to 40%, indicative of conformational changes. It is suggested that this change occurs due to the free energy gain from the ability of the protein to unfold on the less crowded PS surface.