Nanoparticles have received enormous attention as a promising tool to enhance target-specific drug delivery and diagnosis. Various in vitro and in vivo techniques are used to characterize a new system and predict its clinical efficacy. These techniques enable efficient comparison across nanoparticles and facilitate a product optimization process. On the other hand, we recognize their limitations as a prediction tool, due to inadequate applications and overly simplified test conditions. We provide a critical review of in vitro and in vivo techniques currently used for evaluation of nanoparticles and introduce emerging techniques and models that may be used complementarily.