This chapter encompasses applications, fundamentals, instrumentation and future perspectives of desorption electrospray ionization (DESI) mass spectrometry (MS). Since it was first reported in 2004, DESI has paved the way for the development of ambient ionization MS with a wide variety of applications ranging from the analysis of pharmaceutical and illicit drugs to biological lipids and fatty acids. DESI-MS imaging has proven to be one of the most important applications of DESI, yielding comparable data to nonambient MS imaging techniques for tumor classification, virtual fingerprint chemical mapping and many other imaging applications. The development of DESI-MS for such a wide variety of applications has been aided by fundamental studies, which have established a mechanism in which primary DESI droplets first cause analyte dissolution followed by splashing from the surface to produce secondary, sample-containing droplets. A major area that has evolved directly from increased mechanistic understanding is the development of reactive DESI-MS, where reagents are added so as to react with analyte in the secondary droplets to yield better specificity and/or ionization efficiency. In addition to enhancing analytical performance, reactivity in these small secondary droplets often occurs at rates many orders of magnitude faster than bulk-phase reactions. Another developing area of DESI-MS application is its use as an intrasurgical tool to obtain tissue diagnosis including tumor margin assessment. The implementation of DESI-MS for novel applications like these as well as its use in conjunction with a portable mass spectrometer is covered.