For successful translation of targeting nanomedicines from bench to bedside, it is vital to address their most common drawbacks namely rapid clearance and off-target accumulation. These complications evidently originate from a phenomenon called “protein corona (PC) formation” around the surface of targeting nanoparticles (NPs) which happens once they encounter the bloodstream and interact with plasma proteins with high collision frequency. This phenomenon endows the targeting nanomedicines with a different biological behavior followed by an unexpected fate, which is usually very different from what we commonly observe in vitro. In addition to the inherent physiochemical properties of NPs, the targeting ligands could also remarkably dictate the amount and type of adsorbed PC. As very limited studies have focused their attention on this particular factor, the present review is tasked to discuss the best simulated environment and latest characterization techniques applied to PC analysis. The effect of PC on the biological behavior of targeting NPs engineered with different targeting moieties is further discussed. Ultimately, the recent progresses in manipulation of nano-bio interfaces to achieve the most favorite therapeutic outcome are highlighted.