Cell-line tumor spheroids or patient-derived organoids are 3D structures that self-organize when grown in a suitable extracellular matrix. As they mirror in vivo physiology and biology well, these structures have been used as surrogates for clinical trials. This study describes the ability to grow spheroids in NMR-compatible scaffolds that metabolize hyperpolarized [1-13C] pyruvate. These spheroids are also sensitive to AKT inhibition that manifest in significantly decreased lactate production. Histological analysis confirms on-target inhibition, demonstrating that hyperpolarized magnetic resonance spectroscopy can be used to probe treatment response in spheroids that can mimic human disease.