Nutrition represents the strongest lifelong environmental determinant of human health. Modern nutrition research aims at improving health status of individuals through providing new tailored dietary and lifestyle solutions. This concept of personalized nutrition implies a deep knowledge of the complex molecular interplay between genetic makeup and environmental factors including nutrition, metabolism, and food in individual or group of consumers. The discipline of metabolomics is particularly well suited to address this scientific challenge, thanks to its ability to measure the real endpoints of the physiological regulatory processes of living organisms. Furthermore, metabolomics offers the possibility to generate a new generation of biomarkers of health status and nutrition effects at the system level. Nutritional metabolomics employs the metabolic analysis of biological samples mainly using two analytical techniques based on nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) coupled either to gas or liquid chromatography (GC?MS and LC?MS). NMR spectroscopy offers the unique prospect to holistically profile hundreds of metabolites without any preselection. MS methods are also commonly employed for global and targeted profiling with well-adapted sample preparation. MS can generate comprehensive metabolic profiles across chemically diverse classes of compounds including lipids (lipidomics), amino acids biogenic amines, and others. This chapter will review applications and challenges of NMR- and MS-assisted metabolomics for personalized nutrition. © 2013 John Wiley & Sons, Inc. All rights reserved.