Metabolomics is a rapidly growing field of research used in the identification and quantification of the small molecule metabolites within an organism, thereby providing insights into cell metabolism and bioenergetics as well as processes important in clinical medicine, such as disposition of pharmaceutical compounds. It offers comprehensive information about thousands of low-molecular mass compounds (<1500 Da) that represent a wide range of pathways and intermediary metabolism. Because of its vast expansion in the past two decades, mass spectrometry has become an indispensable tool in "omic" analyses. The use of different ionization techniques such as the more traditional electrospray and matrix-assisted laser desorption, as well as recently popular desorption electrospray ionization, has allowed the analysis of a wide range of biomolecules (e.g., peptides, proteins, lipids, and sugars), and their imaging and analysis in the original sample environment in a workup free fashion. An overview of the current state of the methodology is given, as well as examples of application.