Branched-chain amino acids (BCAA) and aromatic AAs (AAA) are associated with increased risk for type 2 diabetes in adults. Studies in youth show conflicting results. We hypothesized that an AA metabolomic signature can be defined to identify youth at risk for β-cell failure and the development of type 2 diabetes. We performed targeted AA metabolomics analysis on 127 adolescents (65 females, 15.5±1.9 years old, Tanner stage II-V) with normal weight or obesity across the spectrum of glycemia, with assessment of AA concentrations by mass spectrometry, at fasting and steady-state (SS) of a hyperinsulinemic-euglycemic clamp, with determination of insulin sensitivity (ISFFM). We measured insulin secretion during a 2-hour hyperglycemic clamp and calculated the disposition index (DIFFM), a measure of β-cell function. Our results showed that Gly and Gln/Glu were lower, whereas BCAA, Tyr and Lys concentrations were higher in the groups with obesity and dysglycemia compared with those with normal weight. Gly and Gln/Glu were positively related to IS and DIFFM, with opposite relationships observed for BCAA, AAA and Lys. We conclude that a metabolic signature of low Gly, low Gln/Glu and elevated BCAA, AAA and Lys may constitute a biomarker to identify youth at risk for β-cell failure.