Mass spectrometry (MS) has emerged as a fundamental technique in metabolomics, owing to its unparalleled sensitivity and specificity in identifying and quantifying metabolites inside intricate biological samples. The development and application of mass spectrometry techniques in metabolomics research have the knowledge to determine the structural and functional value of a biomolecule. Essential subjects encompass the fundamentals of mass spectrometry, advancements in MS technologies like tandem MS and high-resolution MS, and their amalgamation with chromatographic methods such as gas and liquid chromatography techniques. The chapter emphasizes fundamental significance of mass spectrometry in the discovery of biomarkers, metabolic profiling, and pathway analysis in various fields, including illness research, drug development, and environmental investigations. Analyzing ecological changes, pollutant effects, and plant metabolism for better crops, metabolomics is vital in environmental and agricultural settings. Grow stress-resistant plants and boost yields. Integrated with genomics and proteomics, it reveals biological systems and regulatory mechanisms. Metabolomics is crucial to health, agriculture, and environmental management, and biology systems enhance our understanding of diseases and cellular processes. Additionally, contemporary issues, including data interpretation and standardization, as well as prospective developments in MS-based metabolomics, are examined, offering a thorough overview for researchers and practitioners in the domain.