The formation of a protein corona has been considered a pitfall in the clinical translation of nanomedicines. Hence, interdisciplinary studies on corona characterization are critically essential. A deep understanding of the formation of hard and soft protein coronas upon in vivo administration of nanoparticles is vital. The protein corona gives the nanoplatform a new biological identity. Furthermore, the control of and mechanistic understanding of corona formation as it is regulated by the physicochemical properties of nanoparticles is crucial for developing safe nanomedicines. A growing number of analytical techniques have been developed in the past decade for examining NP-protein interactions, contributing to a better understanding of protein corona formation on the surface of nanoparticles. In this Review, we summarize the latest developments in the in vivo and in vitro study of dynamic protein corona formation. Insights derived from techniques used to visualize, quantify, and define protein coronas, as well as the methods for examining the kinetics and structural changes of coronal proteins, are discussed. The potential challenges and future perspectives in the study of protein corona formation and its effects on biological behavior and applications of therapeutic nanomaterials are also provided.