Nanomaterials have a huge potential in research fields from nanomedicine to medical devices. However, surface modifications of nanoparticles (NPs) and thus of their physicochemical properties failed to predict their biological behavior. This requires investigating the “missing link” at the nano-bio interface. The protein corona (PC), the set of proteins binding to the NPs surface, plays a critical role in particle recognition by the innate immune system. Still, in vitro incubation offers a limited understanding of biological interactions and fails to explain the in vivo fate. To date, several reports explained the impact of PC in vitro but its applications in the clinical field have been very limited. Furthermore, PC is often considered as a biological barrier reducing the targeting efficiency of nano vehicles. But the protein binding can actually be controlled by altering PC both in vitro and in vivo. Analyzing PC in vivo could accordingly provide a deep understanding of its biological effect and speed up the transfer to clinical applications. This review demonstrates the need for clarifications on the effect of PC in vivo and the control of its behavior by changing its physicochemical properties. It unfolds the recent in vivo developments to understand mechanisms and challenges at the nano-bio interface. Finally, it reports recent advances in the in vivo PC to overcome and control the limitations of the in vitro PC by employing PC as a boosting resource to prolong the NPs half-life, to improve their formulations and thereby to increase its use for biomedical applications.