Ketoleucine (α-ketoisocaproate) is a novel hyperpolarized substrate for noninvasive metabolic imaging, enabling rapid, high sensitivity detection of branched-chain amino acid flux, a pathway that is aberrant in many diseases including cancer and Alzheimer’s disease. Utilizing NMR Signal Amplification by Reversible Exchange (SABRE) with an 80:20 acetone:water solvent system, we achieved >11% polarization of [1-¹³C]ketoleucine (corresponding to signal enhancement over 230,000-fold at 0.55 T) at 70 mM within 2 min, using parahydrogen as a cheap and fast source of hyperpolarization. A two-stage liquid–liquid extraction and gas stripping protocol removes excess excipients, yielding a biocompatible aqueous solution of [1-¹³C]ketoleucine (79 ± 10 mM). When mixed with Saccharomyces cerevisiae (Baker’s yeast), hyperpolarized [1-¹³C]ketoleucine produced strong, long-lasting signals, where downstream metabolites are observed for >200 s, allowing first-order kinetic modeling of CO₂ and bicarbonate. These cell experiments demonstrate both the biocompatibility and signal strength of SABRE–hyperpolarized KL, establishing KL as a versatile hyperpolarized agent and opening avenues for real-time investigation of metabolic dysregulation in cancer, neurodegenerative disorders, and beyond.