Metabolomics is an emerging component of systems biology that may be a viable strategy for the identification and validation of physiologically relevant biomarkers. Nuclear magnetic resonance (NMR) spectroscopy allows for establishing quantitative data sets for multiple endogenous metabolites without preconception. Sepsis-induced acute lung injury (ALI) is a complex and serious illness associated with high morbidity and mortality for which there is presently no effective pharmacotherapy. The goal of this study was to apply ¹H-NMR based quantitative metabolomics with subsequent computational analysis to begin working towards elucidating the plasma metabolic changes associated with sepsis-induced ALI. To this end, this pilot study generated quantitative data sets that revealed differences between patients with ALI and healthy subjects in the level of the following metabolites: total glutathione, adenosine, phosphatidylserine, and sphingomyelin. Moreover, myoinositol levels were associated with acute physiology scores (APS) (ρ = -0.53, P = 0.05, q = 0.25) and ventilator-free days (ρ = -0.73, P = 0.005, q = 0.01). There was also an association between total glutathione and APS (ρ = 0.56, P = 0.04, q = 0.25). Computational network analysis revealed a distinct metabolic pathway for each metabolite. In summary, this pilot study demonstrated the feasibility of plasma ¹H-NMR quantitative metabolomics because it yielded a physiologically relevant metabolite data set that distinguished sepsis-induced ALI from health. In addition, it justifies the continued study of this approach to determine whether sepsis-induced ALI has a distinct metabolic phenotype and whether there are predictive biomarkers of severity and outcome in these patients.