A key challenge in pharmacology and toxicology is understanding the exposure, fate and effects of drugs and nanomaterials on the body.Nanoparticles arewidely used in topically applied consumer products. For example, titanium dioxide and zinc oxide are used in commercial sunscreen formulations to afford the skin protection from harmful ultraviolet radiation. There are still ongoing safety concerns regarding the long-term toxicity and fate of nanomaterials within complex biological systems with a key question being whether topically applied nanoparticles can penetrate human skin and reach the viable epidermis to cause localized and potentially systemic toxicity under ‘in-use’ conditions. Recent advances in imaging technology have provided us with the tools to map the distribution of native, unlabeled, nanomaterials after application to biological tissue. Another key organ for in situ monitoring is the liver as it is the major site for drug metabolism and excretion in the body. Clearance of drugs from the body can be greatly altered in liver disease, resulting in toxicity and altered effects of the drugs. It is therefore crucial to understand liver functional changes in diseased livers. Recent advances in imaging technology have provided us with insight into mapping the distribution of native, unlabeled, nanomaterials after application to a range of organs within the body. This chapter deals with the use of multiphoton fluorescence microscopy in combination with fluorescence lifetime imaging techniques to assess the safety concerns regarding nanomaterials discussing the key challenges faced.