Animal models such as rats and mice are useful for studying the multiple roles of nitric oxide (NO) in various respiratory disorders. The production of NO is catalyzed by the three isoforms of the enzymes (NO synthases; NOS). Indirect assessment of NOS gene or protein expression only provides partial information on the role of NO in health and lung disease. NO can also be measured in exhaled air by invasive or non-invasive approaches as a direct and quantitative marker of NO production in animal models. Whilst addressing the different methods of exhaled NO analysis in small animals (rats and mice), this review also focuses on the possible clinical implications, and discusses the advantages and limitations of these methods.