This chapter addresses physicochemical properties that affect Nanoparticle (NP) intracellular behavior using Gold NPs (GNPs) as a model system. The main objective is to outline what is known about the effect of GNP size, shape, and surface properties on cellular uptake and intracellular pathway. The authors propose that the entry of GNPs into cells is related to its effectiveness in applications that favor intracellular localization of such GNPs. The authors also discuss how such properties are used to optimize GNP designs for medical applications. Finally, the authors discuss how GNPs may improve disease diagnosis and treatment. Furthermore, how they may be incorporated or used as alternatives to current treatment options is defined.