Significance Untargeted metabolomics is rapidly becoming a mainstream technique to discover biomarkers, to study the effects of interventions, and to discover the function of enzymes. Several thousand metabolites can be detected in a single untargeted metabolomics analysis, but state-of-the-art untargeted metabolomics studies still report up to 40% unidentified metabolites, indicating that large parts of the human metabolome are still unexplored. We identified an uncharacterized class of ubiquitous mammalian metabolites: N -lactoyl-amino acids. Using a powerful combination of proteomics and protein fractionation, we unexpectedly discovered that these metabolites are formed from lactate and amino acids by reversed action of the protease cytosolic nonspecific dipeptidase 2. Our approach to identify unknown metabolites and their biosynthesis has general applicability in the further exploration of the human metabolome.