Surface properties have a significant influence on the performance of biomedical devices. The influence of surface chemistry on the amount and distribution of adsorbed proteins has been evaluated by a combination of atomic force microscopy (AFM) and surface plasmon resonance (SPR). Adsorption of albumin, fibrinogen, and fibronectin was analyzed under static and dynamic conditions, employing self-assembled monolayers (SAMs) as model surfaces. AFM was performed in tapping mode with antibody-modified tips. Phase-contrast images showed protein distribution on SAMs and phase-shift entity provided information on protein conformation. SPR analysis revealed substrate-specific dynamics in each system investigated. When multi-protein solutions and diluted human plasma interacted with SAMs, SPR data suggested that surface chemistry governs the equilibrium composition of the protein layer.