Abstract Nanotechnology is increasingly receiving attention, as the small size of nanocarriers show enhanced properties like high reactivity, strength, surface area, stability, etc. Nanoscale level characterization of nanocarriers presents unique physical, chemical, and biological properties compared to their respective particles at higher scales. To elucidate their physicochemical properties, understanding the nanocarrier structure is important, which greatly influences their behavior in the body at molecular and systemic levels. USFDA has set, or is currently developing, some standard characterization methods for nanomaterials (ISO 22412, ASTME 2859-11). Moreover, to expedite the transition of benchtop efforts to clinically effective products, it is necessary to employ adequate methods to characterize nanocarriers, to correlate their effects and biological consequences, and to predict the therapeutic outcomes in clinical subjects at an early stage of product development. This chapter focuses on various critical quality attributes, methods, and sophisticated instruments available for nanoparticulate characterization for researchers to explore the nanoworld.