After administration of nanoparticles (NP) into biological fluids, the NP may interact with surrounding biomolecules like proteins. Those interactions can then cause protein corona (PC) that affect the physicochemical and biological properties of NP. Numerous studies have been carried out to characterize PC effects in simple and complex biological media but our understanding is far from being complete. Moreover, the variety of methods and tools used for this purpose prevents any robust conclusion and sometimes introduces confusions and uncertainty. Analytical Quality by Design (AQbD), introduced in 2010, is the application of the QbD concept, strongly recommended by FDA, to analytical method development. AQbD aims at optimizing accuracy and robustness of analysis results by identifying and controlling critical quality variables and risk factors over the complete protocol, including biological sample preparation, metrological protocol and statistical analysis.