Ambient ionization techniques, such as nanoDESI and nanoESI, allow for the direct analysis of complex samples under atmospheric pressure with no sample pretreatment. These ionization techniques are utilized for a variety of applications, including lipidomics, online reactions and imaging of small molecules. Nanoelectrospray ionization (nanoESI) is an ionization technique that is similar to electrospray ionization (ESI) but uses smaller sample volumes. NanoESI can be used for complex biological sample analysis and when coupled with online photochemical reactions, such as the Paternò-Büchi (PB) reaction, structural information about lipids can be determined. Likewise, nanoDESI is another ambient ionization technique that employs the ESI mechanism but incorporates online liquid extraction of analytes. This technique is easily incorporated to mass spectrometry imaging (MSI) to provide spatial localization of biomolecules in tissues. Additionally, nanoDESI allows for tunable solvent extraction and online derivatization reactions. These techniques were used to determine structural information of neutral lipids, to image lipids from different developmental stages of lung tissue, and to image and quantify small molecule drugs and metabolites in tissue.