Prostate cancer is the second most common malignancy, and the fifth leading cause of cancer-related death among men, worldwide. A major unsolved clinical challenge in prostate cancer is the ability to accurately distinguish indolent cancer types from the aggressive ones. Reprogramming of metabolism is now a widely accepted hallmark of cancer development, where cancer cells must be able to convert nutrients to biomass while maintaining energy production. Metabolomics is the large-scale study of small molecules, commonly known as metabolites, within cells, biofluids, tissues, or organisms. Nuclear magnetic resonance (NMR) spectroscopy is commonly applied in metabolomics studies of cancer. This chapter provides protocols for NMR-based metabolomics of cell cultures, biofluids (serum and urine), and intact tissue, with concurrent advice for optimal biobanking and sample preparation procedures.