The biological fate of nanomaterials (NMs) is driven by specific interactions through which biomolecules, naturally adhering onto their surface, engage with cell membrane receptors and intracellular organelles. The molecular composition of this layer, called the biomolecular corona (BMC), depends on both the physical-chemical features of the NMs and the biological media in which the NMs are dispersed and cells grow. In this work, we demonstrate that the widespread use of 10% fetal bovine serum in an in vitro assay cannot recapitulate the complexity of in vivo systemic administration, with NMs being transported by the blood. For this purpose, we undertook a comparative journey involving proteomics, lipidomics, high throughput multiparametric in vitro screening, and single molecular feature analysis to investigate the molecular details behind this in vivo/in vitro bias. Our work indirectly highlights the need to introduce novel, more physiological-like media closer in composition to human plasma to produce realistic in vitro screening data for NMs. We also aim to set the basis to reduce this in vitro-in vivo mismatch, which currently limits the formulation of NMs for clinical settings.