The issue of vitamin deficiency persists to be a major health issue worldwide despite the advancements in medicine. At the same time, the effect of marginal vitamin deficiency status on physiological processes is proven. However, general methods such as immune-enzyme and fluorescence analysis, microbiological assays, for example, have limitations in vitamin status assessment and are not able to reliably reflect personal vitamin demand. The potential usefulness of modern metabolomics methods in vitamin status assessment is described in this review. These methods can be used for vitamin metabolites detection as well as for comprehensive metabolic phenotyping that makes them even more valuable.