Abstract: Herein, we present an overview on the current status of the characterization techniques and methodologies used to study the physicochemical descriptors that influence the final clinical performance of a given nanomedicine. The described techniques were selected based on their suitability to operate under relevant “native” conditions that mimic the physiological environment. Special emphasis is placed on those techniques that hold a greater potential to unravel dynamic, structural, and compositional features of soft organic nanomedicines relevant to the ability to bypass biological barriers, and hence allow for the rational design of drug delivery platforms with improved biological output.