We report integrated video and total internal reflection microscopy measurements of protein stabilized 110 nm Au nanoparticles confined in 280 nm gaps in physiological media Measured potential energy profiles display quantitative agreement with Brownian dynamic simulations that include hydrodynamic interactions and camera exposure time and noise effects Our results demonstrate agreement between measured nonspecific van der Waals and adsorbed protein interactions with theoretical potentials Confined, lateral nanoparticle diffusivity measurements also display excellent agreement with piedictions These findings provide a basis to interrogate specific biomacromolecular interactions in similar experimental configurations and to design future improved measurement methods