The new field of nanotoxicology is steadily emerging in parallel with rapid advances made in nanotechnology to evaluate biological impact of intended and non-intended nanomaterial exposure over time as their human applications constantly increase. Over the last decade nanotoxicology methods have mostly relied on in vitro cell-based characterizations that do not account for the complexity of in vivo systems with respect to biodistribution, metabolism, hematology, immunology, and neurological ramifications. Comprehensive in vivo studies addressing the toxicity of nanoscale materials are scarce mainly because the field is still nascent. Efforts in standardizing methodology to study the in vivo safety of these materials are currently undertaken by various government agencies and research organizations. Here, we discuss the need for in vivo nanotoxicity studies, outline some of the important methods, and comment on practical considerations in carrying out such studies.