Due to many unique biological and physicochemical properties, which are only exhibited at the nanoscale, nanomaterials (NMs) have found many applications, including inter alia, in medicines, cosmetics, food, pesticides, textiles, electronics, and construction materials. However, there are concerns over risks posed by NMs to workers, consumers and the environment. The risks emanate from the ability of NMs to translocate from dermal, respiratory, and gastro-intestinal epithelia into the circulatory and lymphatic systems, and ultimately to body tissues and organs, where they can elicit many adverse effects. The adverse effects caused by NMs have been at the center of many studies in environmental toxicology, a field of study that focuses on the effects of toxic agents in humans, the environment, and natural ecosystems. The toxicity of NMs depends on many factors, including size, shape, functional groups, chirality, solubility, reduction–oxidation properties, surface charge, and composition, among others. Therefore, accurate assessment of these physicochemical properties is an absolute imperative. This chapter presents advances, issues, and challenges in the toxicity testing of NMs, including the physicochemical characterization of NMs in both in vitro and in vivo systems as well as in the environment. Moreover, the chapter presents the challenges and recent advances in in vitro , in vivo , and in silico toxicity assessment of NMs.