Inhalation of gases, vapors and can cause a wide range of adverse health effects, ranging from simple irritation to systemic diseases. The large number of chemicals and complex mixtures present in indoor and outdoor air coupled with the introduction of new materials such as nanoparticles and nanofibers, is an area of growing concern for human health. Animal-based assays have been used to study the toxic effects of chemicals for many years. However, even so, very little is known about the potential toxicity of the vast majority of inhaled chemicals. As well as new or refined OECD test guidelines, continuing scientific developments are needed to improve the process of safety evaluation for the vast number of chemicals and inhaled materials. Although studying the toxic effects of inhaled chemicals is more challenging, promising in vitro exposure techniques have been recently developed that offer new possibilities to test biological activities of inhaled chemicals under biphasic conditions at the air liquid interface. This chapter gives an overview of inhalation toxicology as well as focusing on the potential application of in vitro methods for toxicity testing of airborne pollutants.