Hyperglycemia causes diabetic nephropathy, a condition for which there are no specific diagnostic markers that predict progression to renal failure. Here we describe a multiplatform metabolomic analysis of urine from individuals with type 2 diabetes, collected before and immediately following an intravenous glucose load. We used targeted nuclear magnetic resonance spectroscopy (NMR), liquid chromatography - mass spectrometry (LC-MS) and gas chromatography - MS (GC-MS) to identify markers of hyperglycemia. Following optimization of data normalisation and statistical analysis, we identified a reproducible NMR and LC-MS based urine signature of hyperglycemia. Hyperglycaemia was associated with significant increases of alanine, alloisoleucine, isoleucine, leucine, N-isovaleroylglycine, valine, choline, lactate and taurine and decreases of arginine, gamma-aminobutyric acid, hippurate, suberate and N-acetylglutamate. GC-MS analysis identified a number of metabolites differentially present in post-glucose versus baseline urine, but these could not be identified using current metabolite libraries. This analysis is an important first step towards identifying biomarkers of early-stage diabetic nephropathy.