In modern nutrition research, mass spectrometry has developed into a tool to assess health, sensory as well as quality and safety aspects of food. In this review, we focus on health-related benefits of food components and, accordingly, on biomarkers of exposure (bioavailability) and bioefficacy. Current nutrition research focuses on unraveling the link between dietary patterns, individual foods or food constituents and the physiological effects at cellular, tissue and whole body level after acute and chronic uptake. The bioavailability of bioactive food constituents as well as dose-effect correlations are key information to understand the impact of food on defined health outcomes. Both strongly depend on appropriate analytical tools to identify and quantify minute amounts of individual compounds in highly complex matrices--food or biological fluids--and to monitor molecular changes in the body in a highly specific and sensitive manner. Based on these requirements, mass spectrometry has become the analytical method of choice with broad applications throughout all areas of nutrition research. The current review focuses on selected areas of application: protein and peptide as well as nutrient and metabolite analysis.