Allergic airway inflammation is commonly associated with enhanced exhaled nitric oxide (ENO) in both humans and mice. Since mouse models are being used to understand various mechanisms of asthma, a non-invasive simple and reproducible method to determine ENO in mice is required for serial non-terminal assessment that can be used independent of environmental situations where the ambient air contains substantial amounts of NO as a contaminant. The aim of this study was to non-invasively measure ENO in individual mice and to test its utility as a marker of airway inflammation (AI) in different models of allergic airway inflammation. We modified the existing ENO measuring methods by incorporating flushing and washout steps that allowed simple but reliable measurements under highly variable ambient NO conditions (1 - 100 ppb). This method was used to serially follow ENO in acute and chronic models of allergic airway inflammation in mice. ENO was reproducibly measured by this modified method, and was positively correlated to AI in both acute and chronic models of asthma, but was not independently related to airway remodeling. Resolution of AI and other related parameters in dexamethasone treated mice resulted in reduction of ENO, further confirming this association. Restriction of allergen challenge to pulmonary but not nasal airways was associated with a smaller increase in ENO compared to allergen challenge to both. Hence, ENO can now be reliably measured in mice independent of ambient NO levels, and is a valid biomarker for airway inflammation. However, nasal and pulmonary airways are likely to be independent sources of ENO and any results must be interpreted as such.