Tubulointerstitial fibrosis is a characteristic hallmark of chronic kidney disease (CKD). Metabolic perturbations in cellular energy metabolism contribute to the pathogenesis of CKD, but the chemical contributors remain unclear. The aim of this investigation was to use two dimensional 1H-nuclear magnetic resonance (2D-COSY) metabolomics to identify the chemical changes of kidney fibrogenesis. An in vitro transforming growth factor-β1 (TGF-β1)-induced model of kidney fibrogenesis with human kidney-2 (HK-2) proximal tubular epithelial cells (PTEC) was used. The model was validated by assaying for various pro-fibrotic molecules, using quantitative PCR and Western blotting. 2D-COSY was performed on treated cells. Morphological and functional changes characteristic of tubulointerstitial fibrosis were confirmed in the model; expression of fibronectin, collagen type IV, smooth muscle actin, oxidative stress enzymes increased (p < 0.05). NMR metabolomics provided evidence of altered metabolite signatures associated with glycolysis and glutamine metabolism, with decreased myo-inositol and choline, and metabolites of the oxidative phase of the pentose phosphate pathway with increased glucose and glucuronic acid. The altered PTEC cellular metabolism likely supports the rapid fibrogenic energy demands. These results, using 2D-COSY metabolomics, support development of a biomarker panel of fibrosis detectable using clinical magnetic resonance spectroscopy to diagnose and manage CKD.