Acute myeloblastic leukemia (AML) is one of the most common and life-threatening forms of leukemia. Treatment remains challenging due to its high heterogeneity, drug resistance, and metabolic flexibility. Targeting specific metabolic pathways has emerged as a promising therapeutic approach. The ability to monitor treatment response is crucial for disease management. Here, we utilized hyperpolarized ¹³C nuclear magnetic resonance (NMR) spectroscopy to evaluate the therapeutic effects of 2-deoxy-D-glucose (2-DG), a glucose analog known to inhibit glycolysis and induce cell death in leukemic cell lines. Hyperpolarized ¹³C NMR spectroscopy, biochemical assays, and respirometry were used to assess the metabolic effects of 2-DG treatment at various concentrations on the AML cell line ML-1 in vitro. Significant metabolic alterations were observed following 2-DG treatment at 2 mM and 5 mM for 24 h, as revealed by multiple analytical approaches. The concentration-dependent effects of 2-DG treatment were clearly detected using hyperpolarized NMR, demonstrating substantial inhibition of glycolytic pathways in ML-1 cells. This study supports the potential of 2-DG for enhancing chemosensitivity in AML treatment and highlights hyperpolarized NMR as a valuable tool for therapy evaluation.