Identification of highly aggressive tumors from benign phenotypes is an important diagnostic need, with increased expression of hypoxia-inducible factor-1α (HIF-1α) as one factor linked to tumor progression and treatment response. HIF-1α stabilization is closely linked to extracellular pH (pHe) through the regulation of glycolysis, proton transporters and vascular endothelial growth factor (VEGF). Magnetic resonance imaging (MRI)-based pHe measurement has shown promise in differentiating tumor phenotypes. In this study, we evaluated a new protocol designed to enhance tumor acidosis by administering glucose prior to pHe measurements in orthotopic human breast cancer xenografts. To demonstrate the utility of this approach, we used human triple-negative breast cancer (TNBC) MDA-MB-231 cells that were either wild type (231-WT) or genetically engineered to stably express short hairpin RNA (shRNA) against HIF-1α (231-HIF-1α-shRNA) or engineered to overexpress VEGF (231-VEGF). Iopamidol was administered for chemical exchange saturation transfer (CEST) MRI pHe mapping. We observed enhanced differentiation in the pHe maps following glucose administration, with mean pHe of 6.1 ± 0.12 (231-WT), 6.3 ± 0.04 (231-VEGF) and 6.58 ± 0.04 (231-HIF-1α-shRNA). Without glucose stimulation, the corresponding values were 6.31 ± 0.04 (231-WT), 6.30 ± 0.07 (231-VEGF), and 6.55 ± 0.04 (231-HIF-1α-shRNA). These findings were validated by immunoblotting and immunohistochemistry. Collectively our data demonstrate that CEST MRI pHe mapping can effectively differentiates tumors with low HIF-1α expression and that glucose stimulation enhances this differentiation, offering a valuable tool for improved tumor characterization.