Metabolite analysis is essential for understanding the biochemical processes and pathways that sustain life, providing insights into the complex interactions within cellular systems and clinical examinations. This review explores recent applications of nuclear magnetic resonance (NMR) spectroscopy in metabolite studies. Various methods enhancing analytical accuracy for metabolome profiling and metabolic pathway studies, including spectral simplification techniques, quantitative NMR, high-resolution MAS NMR, and isotopic labeling, are discussed. The application of NMR in in situ and in vivo studies is also covered, highlighting in-cell NMR and in vivo MRS techniques. Last but not least, we discuss recent advancements in NMR hyperpolarization, with a focus on dynamic nuclear polarization (DNP), chemically induced dynamic nuclear polarization (CIDNP), para-hydrogen-induced polarization (PHIP), and signal amplification by reversible exchange (SABRE). These advancements offer significant potential for enhancing the sensitivity and accuracy of metabolite studies and are expected to further deepen the study and understanding of metabolites and metabolic pathways.