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Background. Urolithiasis is the process of stone formation in the urinary tract. Its etiology is only partly known, and efficient therapeutic approaches are currently lacking. Metabolomics is increasingly used in biomarkers discovery for its ability to identify mediators of relevant (patho)physiological processes. Amino acids may be involved in kidney stone formation. The aim of the present study was to investigate the presence of an amino acid signature in stone former urine through a targeted metabolomic approach. Methods. A panel of 35 amino acids and derivatives was assessed in urines from 15 stone former patients and 12 healthy subjects by UPLC-MS. Partial Least Squares Discriminant Analysis (PLS-DA) was used to define amino acid profiles of cases and controls. Results and Discussion. Our approach led to the definition of a specific amino acid fingerprint in people with kidney stones. A urinary amino acid profile of stone formers was characterized by lower levels of α-aminobutyric acid, asparagine, ethanolamine, isoleucine, methionine, phenylalanine, serine, tryptophan, and valine. Metabolomic analysis may lend insights into the pathophysiology of urolithiasis and allow tracking this prevalent condition over time. [ABSTRACT FROM AUTHOR]