Quality-by-Design (QbD) guidance is a risk-based and proactive approach to drug development proposed in the mid 2000s and now widely used in the pharmaceutical field in compliance with the ICH Q8-Q11 guidelines. Analytical Quality by Design (AQbD), introduced in 2010, is the adaptation of the QbD paradigm for the development of analytical methods. AQbD aims at optimizing the accuracy and robustness of analysis results by identifying and controlling critical analytical variables and method parameters over the entire protocol, including biological sample preparation, measurement technology and statistical analysis. Nevertheless, much remains to be done for a clear understanding and an efficient implementation of this new paradigm in practice. The first objective of this review is to propose a global clarification of the Analytical Quality by Design approach by reviewing its terminology and steps and by clarifying its relationships with the well-established QbD paradigm and ICH guidelines. Two new templates of documents have been proposed: a form designed for the definition of the analytical target profile and a connection matrix between expected metrological properties and analytical attributes. Finally, the open challenges in the characterization of nano-enabled medicinal products are examined from the AQbD angle.