Synthetic as well as cell-based nanocarriers have come into great consideration for treating neurodegenerative diseases as well as other cerebral conditions. How well the brain-targeting delivery of drugs happens using Nano formulations is hugely determined by the physicochemical parameters such as size, shape, hydrophobicity, elasticity, and charge/chemistry/morphology at the surface of the drug nanocarrier, which determines their mode of interaction with living systems. One of the key determinants of their in vivo behavior is the protein corona formation, which governs nanoparticle recognition, circulation, and biodistribution. It is important to understand the biological matrices and cell culture compositions involved in protein corona formation in order to design efficient nanomedicines. In addition, characterization of nanocarriers in complex biological environments poses specific challenges, and advanced analytical methods need to be developed and used. This review discusses the types and properties of brain-targeted nanocarriers, there in vivo interactions, and the characterization methods employed for them. We also discuss the strengths and weaknesses of existing analytical tools, the difficulties in applying these methods in a Good Manufacturing Practice (GMP) setting, and the promise of orthogonal complementary characterization methods. By overcoming these challenges, this review will offer the insights into how the translational value of nanomedicines in brain disorders can be improved.