The field of metabolomics has witnessed an exponential growth in the last decade driven by important applications spanning a wide range of areas in the basic and life sciences and beyond. Mass spectrometry in combination with chromatography and nuclear magnetic resonance are the two major analytical avenues for the analysis of metabolic species in complex biological mixtures. Owing to its inherent significantly higher sensitivity and fast data acquisition, MS plays an increasingly dominant role in the metabolomics field. Propelled by the need to develop simple methods to diagnose and manage the numerous and widespread human diseases, mass spectrometry has witnessed tremendous growth with advances in instrumentation, experimental methods, software, and databases. In response, the metabolomics field has moved far beyond qualitative methods and simple pattern recognition approaches to a range of global and targeted quantitative approaches that are now routinely used and provide reliable data, which instill greater confidence in the derived inferences. Powerful isotope labeling and tracing methods have become very popular. The newly emerging ambient ionization techniques such as desorption ionization and rapid evaporative ionization have allowed direct MS analysis in real time, as well as new MS imaging approaches. While the MS-based metabolomics has provided insights into metabolic pathways and fluxes, and metabolite biomarkers associated with numerous diseases, the increasing realization of the extremely high complexity of biological mixtures underscores numerous challenges including unknown metabolite identification, biomarker validation, and interlaboratory reproducibility that need to be dealt with for realization of the full potential of MS-based metabolomics. This chapter provides a glimpse at the current status of the mass spectrometry-based metabolomics field highlighting the opportunities and challenges.