Evaluation of renal dysfunction includes estimation of glomerular filtration rate (eGFR) as the initial step and subsequent laboratory testing. We hypothesized that combined analysis of serum creatinine, myo-inositol, dimethyl sulfone, and valine would allow both assessment of renal dysfunction and precise GFR estimation. Bio-banked sera were analyzed using nuclear magnetic resonance spectroscopy (NMR). The metabolites were combined into a metabolite constellation (GFR_NMR) using n = 95 training samples and tested in n = 189 independent samples. Tracer-measured GFR (mGFR) served as a reference. GFR_NMR was compared to eGFR based on serum creatinine (eGFR_Crea and eGFR_EKFC), cystatin C (eGFR_Cys-C), and their combination (eGFR_Crea-Cys-C) when available. The renal biomarkers provided insights into individual renal and metabolic dysfunction profiles in selected mGFR-matched patients with otherwise homogenous clinical etiology. GFR_NMR correlated better with mGFR (Pearson correlation coefficient r = 0.84 vs. 0.79 and 0.80). Overall percentages of eGFR values within 30% of mGFR for GFR_NMR matched or exceeded those for eGFR_Crea and eGFR_EKFC (81% vs. 64% and 74%), eGFR_Cys-C (81% vs. 72%), and eGFR_Crea-Cys-C (81% vs. 81%). GFR_NMR was independent of patients’ age and sex. The metabolite-based NMR approach combined metabolic characterization of renal dysfunction with precise GFR estimation in pediatric and adult patients in a single analytical step.