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