Dysregulation of cellular metabolism is a key feature of major diseases such as diabetes and cancer. Monitoring cellular metabolic alterations is of critical importance for improved understanding of disease progression and the development of novel therapeutics. Carbon-13 magnetic resonance spectroscopy (13C MRS), which can detect and quantitate metabolites in a non-invasive manner, is well suited for clinical and pre-clinical metabolic studies. The low natural abundance of 13C allows one to trace exogenously administered 13C-labeled substrates by MRS and which enables the study of complex metabolic pathways in living subjects. However, the low sensitivity of 13C MRS limits the number of metabolites that can be detected with this technique. Hyperpolarized (HP) 13C MRS using dissolution dynamic nuclear polarization (d-DNP) is a novel metabolic imaging technique which allows real-time monitoring of the dynamics of in vivo metabolism by temporarily increasing the signal intensity more than 10,000-fold over conventional 13C MRS. This thesis is focused on the biomedical applications of HP 13C MRS, particularly to investigate in vivo hepatocellular energy metabolism in rodents and metabolic changes in active immune cells. Customized MRI/NMR probes and associated acquisition sequences were designed and implemented to improve the sensitivity and throughput of the HP MR experiments. A HP 13C MRS protocol for the sensitive in vivo detection of hepatic pyruvate metabolism was developed. HP [1-13C]pyruvate was infused at progressively decreasing doses to healthy rats under different nutritional conditions via a rapid automated infusion. It was shown that liver metabolism can be measured in vivo with HP [1-13C]pyruvate administered at near-physiological levels. While conversion to HP bicarbonate was detected in the liver of fasted rats, treatment with the phosphoenolpyruvate carboxykinase inhibitor 3-mercaptopicolinate resulted in significantly lower levels compared to fed rats, which supports the notion that hepatic gluconeogenic metabolism can be directly probed in vivo with HP pyruvate. Additionally, real-time in vivo hepatocellular glutamine and pyruvate metabolism were investigated in hepatocellular carcinoma (HCC) induced in liver receptor homolog-1 (LRH-1) knockout mice. The influence of LRH-1 on glutaminase activity was monitored through the conversion of HP [5-13C]glutamine to [5-13C]glutamate, and the impact of LRH-1 deletion on the citric acid cycle could be investigated by conversion of HP [1-13C]pyruvate to four-carbon mitochondrial metabolites. The results demonstrate the potential of HP 13C MRS as a non-invasive method for understanding the role of oncogenic mutations in nuclear receptors in the metabolic reprogramming of cancer cells. Several other 13C-labeled substrates were studied, including [1-13C]cysteine and γ-glutamyl[1-13C]glycine, a novel probe to detect gamma-glutamyl transferase (GGT) activity. The metabolic changes in T cells upon activation was investigated using HP [1-13C]cysteine as a potential selective probe of active T cells and HP γ-glutamyl[1-13C]glycine to detect increased GGT expression by T cells in the active state. This work includes the first report on applications of HP 13C MRS in T cells and demonstrates the feasiblity to study the immune response with this technique. In conclusion, this work shows the potential of HP 13C MRS to investigate in vivo liver metabolism with administered 13C-labeled