Rationale: Airway obstruction in patients with asthma is associated with airway dysfunction and inflammation. Objective measurements including sputum analysis can guide therapy, but this is often not possible in typical clinical settings. Metabolomics is the study of molecules generated by metabolic pathways. We are developing a noninvasive means of monitoring asthma status using metabolomics and urine sampling. We hypothesize that airway dysfunction and inflammation in an animal model of asthma would produce unique patterns of urine metabolites measured by multivariate statistical analysis of high-resolution ¹H nuclear magnetic resonance spectroscopy (NMR) data. Methods: Five groups of guinea pigs were studied: control, control treated with dexamethasone, sensitized (ovalbumin i.p.), sensitized and challenged (ovalbumin i.p. + ovalbumin aerosol), and sensitized-challenged with dexamethasone. Airway hyperreactivity (AHR) to histamine (i.v.) and inflammation were measured. Multivariate statistical analysis of NMR spectra based on a library of known urine metabolites was performed using partial least squares discriminant analysis (PLS-DA). In addition, the raw NMR spectra exported as xy-trace data underwent linear discriminant analysis (LDA). Results: Challenged guinea pigs developed AHR and increased inflammation compared with sensitized or control. Dexamethasone significantly improved AHR. Using concentration differences in metabolites, PLS-DA could discriminate challenged animals with 90% accuracy. Using only 3-4 regions of the NMR spectra, LDA-based classification demonstrated 80-90% separation of the animal groups. Conclusion: Urine metabolites correlate with airway dysfunction in an asthma model. Urine NMR analysis is a promising, noninvasive technique for monitoring asthma in humans.