Metabolic phenotyping comprises the quantification of endogenous meta- bolites in biofluids, cells, and tissues. It provides insights into normal as well as aberrant metabolic pathways and biological processes, which is important for the understanding of disease phenotypes. It also allows the identification of biological markers, which can serve as early disease indicators and therapeutic markers for the evaluation of treatment effects. As metabolic markers are not species-restricted, the concept of metabolic phenotyping is highly applicable for translational research. Species independence allows the use of established animal and cell culture models for various diseases within a preclinical context. However, differences in the metabolic set-up of study organisms compared to humans needs to be taken into consideration to prevent misleading conclusions from otherwise valid experimental designs. To deter- mine species-related metabolic differences, a targeted metabolomics approach was applied using a mass spectrometry platform for the quantification of a predefined set of endogenous metabolites, i.e. amino acids, biogenic amines, phosphatidylcholines, sphingomyelins, hexoses, steroid hormones and others. Results from this species comparison on the metabolic level will be presented. Overall, the validity of metabolic phenotyping will be demonstrated, despite or even because of species-dependent characteristics. It has the potential to explain why findings in animal models cannot always be directly translated into clinical settings and might, therefore, facilitate the establishment of suitable models of disease.