Lipids have essential roles in several biological processes, including normal cell functioning as well as in the onset of diseases. Renewed interest in lipid research has been largely fueled by emerging evidence linking the augmenting of corporal adipose tissue in humans with metabolic diseases and cancer. Imaging mass spectrometry techniques including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) are analytical methods quite suitable for the investigation of compounds in biological samples, providing an enormous amount of information regarding their chemical composition and spatial distribution with no need for prior labeling. Particularly, MALDI-TOF imaging mass spectrometry (IMS) has evolved into a very versatile technique capable of analyzing, at molecular level, a wide range of compounds, including lipids. This review presents a survey of recent work conducted mainly in our laboratory on full protocol development for lipid analysis by MALDI-TOF IMS of biological samples, with emphasis on sample collection and preparation, matrix selection and deposition, and typical analysis condition settings.