Urine is produced from blood by the kidneys that extract soluble wastes, excess water and several other compounds from the bloodstream. Urine is readily available in high volumes and can be collected noninvasively. Urine is chemically complex; containing both endogenous and exogenous compounds, and reflects imbalances of several biochemical pathways, which can be related to (patho)physiological conditions. In addition to a metabolic state; urine also reflects gut microbial metabolic activities and short-term food consumption. As inter- and intra-individual variations in urine samples are remarkable, large sample sizes together with the use of biologically optimal normalisation method for absolute urine metabolite concentrations are needed to be able to reliably detect metabolite alterations related to biological conditions.