Metabolomics is the study of changes in the metabolic status of an organism as a consequence of drug treatment, environmental influences, nutrition, lifestyle, genetic variations, toxic exposure, disease, stress, etc, through global or comprehensive identification and quantification of every single metabolite in a biological system. Since most chronic diseases have been demonstrated to be linked to nutrition, nutritional metabolomics has great potential for improving our understanding of the relationship between disease and nutritional status, nutrient, or diet intake by exploring the metabolic effects of a specific food challenge in a more global manner, and improving individual health. In particular, metabolite profiling of biofluids, such as blood, urine, or feces, together with multivariate statistical analysis provides an effective strategy for monitoring human metabolic responses to dietary interventions and lifestyle habits. Therefore, studies of nutritional metabolomics have recently been performed to investigate nutrition-related metabolic pathways and biomarkers, along with their interactions with several diseases, based on animal-, individual-, and population-based criteria with the goal of achieving personalized health care in the future. This article introduces analytical technologies and their application to determination of nutritional phenotypes and nutrition-related diseases in nutritional metabolomics.