Since the introduction of desorption electrospray ionization (DESI) mass spectrometry (MS), ambient MS methods have seen increased use in a variety of fields from health to food science. Increasing its popularity in metabolomics, ambient MS offers limited sample preparation, rapid and direct analysis of liquids, solids, and gases, in situ and in vivo analysis, and imaging. The metabolome consists of a constantly changing collection of small (<1.5KDa) molecules. These include endogenous molecules that are part of primary metabolism pathways, secondary metabolites with specific functions such as signaling, chemicals incorporated in the diet or resulting from environmental exposures, and metabolites associated with the microbiome. Characterization of the responsive changes of this molecule cohort is the principle goal in any metabolomics study. With adjustments to experimental parameters, metabolites across a range of chemical and physical properties can be selectively desorbed and ionized and subsequently analyzed with increased speed and sensitivity. This review covers the broad applications of a variety of ambient MS techniques in four primary fields in which metabolomics is commonly employed.