A rapid resolution liquid chromatography-tandem mass spectrometry analysis was applied to human urine samples to discover new molecular biomarkers of the alteration of psychophysical well-being due to the environmental, physiological and confinement stress conditions achieved in the MARS500 project. Urine samples of crew members were submitted to metabolomic studies by means of separative techniques coupled with mass spectrometric techniques and data analysis. More than 2,000 metabolite features were found in each comparison, and more than 150 metabolites were identified for every differential analysis. Significant modifications in eight metabolites were shared by the subjects under study.