The recent guidance on "Safety Testing of Drug Metabolites" issued by the U.S. Food and Drug Administration, Center for Drug Evaluation and Research (CDER) has highlighted the importance of identifying and characterizing drug metabolites as early as possible in drug discovery and development. Furthermore, upon identifying significant circulating metabolites in human plasma, it has become important to demonstrate that these metabolites are present at an equal or greater exposure level (area under the curve, AUC) in any one of the preclinical species used in safety testing. Frequently, synthetic standards of metabolites are not available, and hence, obtaining their AUC values can be a challenge. In this report, we demonstrate how combinations of nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography/ultraviolet/mass spectrometry (LC/UV/MS), and plasma pooling methods were used to obtain reliable AUC values of metabolites present in the plasma of preclinical species from short-term safety studies. Plasma pooling methods were compared to the traditional approaches of obtaining quantitative information on the levels of circulating metabolites in preclinical species. The exposure values obtained via sample pooling were comparable to those obtained by traditional methods of analyzing samples individually. In the absence of synthetic chemical standards, calculations of AUC values of metabolites, using either sample pooling or traditional approaches, were achieved through the use of UV detectors. In cases where the UV properties of metabolites were significantly different from their parent compounds, NMR was used as a quantitative tool to obtain exposure values. NMR was found to be useful in quantitating biologically produced metabolites, which could subsequently be used as reference compounds for further quantitative studies. The limitations of UV detectors to obtain exposure estimates are discussed. A practical solution is presented that will enable us to obtain a quantitative assessment of metabolite exposure in humans and coverage in toxicology species, hence, circumventing the use of radiolabeled compounds or authentic chemically synthesized standards of metabolites.