Here, we propose a new theoretical framework that enables the use of Differential Dynamic Microscopy (DDM) in fluorescence imaging mode to quantify in situ protein adsorption onto nanoparticles (NP) while simultaneously monitoring for NP aggregation. We use this methodology to elucidate the thermodynamic and kinetic properties of the protein corona (PC) in vitro and in vivo. Our results show that protein adsorption triggers particle aggregation over a wide concentration range and that the formed aggregate structures can be quantified using the proposed methodology. Protein affinity for polystyrene (PS) NPs was observed to be dependent on particle concentration. For complex protein mixtures, our methodology identifies that the PC composition changes with the dilution of serum proteins, demonstrating a Vroman effect never quantitatively assessed in situ on NPs. Finally, DDM allows monitoring of the evolution of the PC in vivo. Our results show that the PC composition evolves significantly over time in zebrafish larvae, confirming the inherently dynamic nature of the PC. The performance of the developed methodology allowed to obtain quantitative insights into nano-bio interactions in a vast array of physiologically relevant conditions that will serve to further improve the design of nanomedicine. This article is protected by copyright. All rights reserved.