Metabolomics is the systematic study of all the metabolites present within a biological system, which consists of a mass of molecules, having a variety of physical and chemical properties and existing over an extensive dynamic range in biological samples. Diverse analytical techniques are needed to achieve higher coverage of metabolites. The application of mass spectrometry (MS) in metabolomics has increased exponentially since the discovery and development of electrospray ionization and matrix-assisted laser desorption ionization techniques. Significant advances have also occurred in separation-based MS techniques (gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, capillary electrophoresis-mass spectrometry, and ion mobility-mass spectrometry), as well as separation-free MS techniques (direct infusion-mass spectrometry, matrix-assisted laser desorption ionization-mass spectrometry, mass spectrometry imaging, and direct analysis in real time mass spectrometry) in the past decades. This review presents a brief overview of the recent advanced MS techniques and their latest applications in metabolomics. The software/websites for MS result analyses are also reviewed.