Water-assisted laser desorption/ionization mass spectrometry (WALDI-MS), also known as SpiderMass, is an emerging ambient ionization technique for in vivo and real-time analysis. It employs a remote infrared (IR) laser tuned to excite the most intense vibrational band (O-H) of water. The water molecules act as an endogenous matrix leading to the desorption/ionization of a variety of biomolecules from tissues, particularly metabolites and lipids. WALDI-MS was recently advanced into an imaging modality for ex vivo 2D sections and 3D in vivo real-time imaging. Here, we describe the methodological aspects for performing 2D and 3D imaging experiments with WALDI-MSI and the parameters for optimizing the image acquisition.