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Protein adsorption to nanoparticles (NPs) is a key prerequisite to understand NP–cell interactions. While the layer thickness of the protein corona has been well characterized in many cases, the absolute number of bound proteins and their exchange dynamics in body fluids is difficult to assess. Here we measure the number of molecules adsorbed to sulfonate (PSOSO₃H) and carboxyl-(PSCOOH) polystyrene NPs using fluorescence correlation spectroscopy. We find that the fraction of molecules bound to NPs falls onto a single, universal adsorption curve, if plotted as a function of molar protein-to-NP ratio. The adsorption curve shows the build-up of a strongly bound monolayer up to the point of monolayer saturation (at a geometrically defined protein-to-NP ratio), beyond which a secondary, weakly bound layer is formed. While the first layer is irreversibly bound (hard corona), the secondary layer (soft corona …