In biological fluids, proteins bind to particles, forming so-called protein coronas. Such adsorbed protein layers significantly influence the biological interactions of particles, both in vitro and in vivo. The adsorbed protein layer is generally described as a two-component system comprising "hard" and "soft" protein coronas. However, a comprehensive picture regarding the protein corona structure is lacking. Herein, we introduce an experimental approach that allows for in situ monitoring of protein adsorption onto silica microparticles. The technique, which mimics flow in vascularized tumors, combines confocal laser scanning microscopy with microfluidics and allows the study of the time-evolution of protein corona formation. Our results show that protein corona formation is kinetically divided into three different phases: phase 1, proteins irreversibly and directly bound (under physiologically relevant conditions) to the particle surface; phase 2, irreversibly bound proteins interacting with preadsorbed proteins, and phase 3, reversibly bound "soft" protein corona proteins. Additionally, we investigate particle-protein interactions on low-fouling zwitterionic-coated particles where the adsorption of irreversibly bound proteins does not occur, and on such particles, only a "soft" protein corona is formed. The reported approach offers the potential to define new state-of-the art procedures for kinetics and protein fouling experiments.