Functional gold nanoparticles have emerged as a cornerstone in targeted drug delivery, imaging, and biosensing. Their stability, distribution, and overall performance in biological systems are largely determined by their interactions with molecules in biological fluids as well as the biomolecular layers they acquire in complex environments. However, real-time tracking of how biomolecules attach to colloidal nanoparticles, a critical aspect for optimizing nanoparticle function, has proven to be experimentally challenging. To address this issue, we present a depolarized forward light scattering (DFLS) method that measures rotational relaxation constants. In DFLS, optically anisotropic nanoparticles are illuminated with linearly polarized light and the forward light scattering is analyzed in a cross-polarized configuration. We demonstrate the application of DFLS to characterize various functional coatings, analyze biomolecular binding kinetics to gold nanoparticles, and determine specific protein adsorption affinity constants. Our results indicate that DFLS offers a powerful approach to studying nanoparticle-biomolecule interactions in complex environments such as bodily fluids, thereby opening new pathways for advancements in nanomedicine and the optimization of nanoparticle-based drug delivery systems.