Hyperpolarized (HP) [1-¹³C]pyruvate nuclear magnetic resonance (NMR) spectroscopy was employed to investigate tumor energy metabolism in tiny multicellular spheroids, serving as a model of early-phase tumorigenesis in vivo. A three-dimensional static culture of murine squamous cell carcinoma (SCCVII)cells formed uniform smaller multicellular spheroids (~ 150 μm in diameter), without hypoxic or necrotic cores, yet these spheroids exhibited resistance to anti-tumor drugs. HP [1-¹³C]pyruvate NMR spectroscopy of SCCVII spheroids revealed an increased conversion of pyruvate to lactate compared to monolayer cultures, indicating enhanced aerobic glycolysis in the aggregated cells. Additionally, HP spectroscopy differentiated the degree of aerobic glycolysis in human prostate tumorspheroids―DU145 (~ 120 μm) and PC-3 (~ 230 μm)―as evidenced by the upregulation of genes associated with lactate production and cellular transport. The Lac/Pyr ratio among spheroids correlated with those observed in biopsy samples of corresponding malignant tumors grown in mice. These findings suggest that HP [1-13C]pyruvate NMR spectroscopy may serve as a metabolic biomarker for early-phase tumorigenesis in vivo.