Mass Spectroscopy Imaging (MSI) has emerged as an enabling technique to provide insight into the molecular entities within cells, tissues and whole-body samples and to understand inherent complexities within biological metabolomes. In Mass Spectrometry Imaging of Small Molecules: Methods and Protocols, experts in the MSI field present techniques for 2D and 3D visualization and quantification of a wide array of small molecular species present in biologically relevant samples. Chapters provide detailed operational instructions from sample preparation to method selection, from comparative quantification to structural identification and from data collection to visualization of small molecule mapping in complex samples. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Mass Spectrometry Imaging of Small Molecules: Methods and Protocols aims to bring the rapidly maturing methods of metabolic imaging to life science researchers and to minimize technical intimidation in adapting new technological platforms in biological research.