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Deliberate and accidental exposure of the ecosystem including humans to nanoparticles becomes inevitable as nanomaterials are increasingly used. In biological fluids, biomolecules associate with nanoparticles, leading to the formation of a dynamic biomolecule “corona” that critically defines the biological identity of the particle. As the bio-physical properties of such a decorated particle often differ significantly from those of the formulated particle a detailed characterization of nanoparticles in biological environments becomes increasingly important though, nevertheless also technically challenging. Here, we introduce experimental methods currently employed for nanoparticle characterization, present examples underlining the complexity of the nano-bio interface, and discuss the need for further technical and conceptual developments. A deep and mechanistic bio-physical understanding of the nano-bio interface is a challenge but also fundamental prerequisite for future applications in nanobiology, nanomedicine and nano(eco)toxicology.