Matrix-assisted laser desorption/ionization (MALDI)-imaging mass spectrometry (IMS, also referred to as mass spectrometry imaging [MSI]) enables visualization of the distribution of biomolecules with varied and vast structures in tissue sections. This emerging imaging technique was initially developed as a tool for protein imaging; however, the number of studies reporting imaging of small organic molecules has recently increased. IMS is an effective technique for the visualization of endogenous small metabolites, especially lipids, facilitated by the unique advantages of mass spectrometry-based molecular detection. Despite the promising capability of MALDI-IMS for imaging small metabolites, this technique still has several issues, especially in spatial resolution. One of the critical limitations of the spatial resolution of MALDI-IMS is the size of the organic matrix crystal and the analyte migration during the matrix application process. To overcome these problems, we reported a nanoparticle (NP)-assisted laser desorption/ionization (nano-PALDI)-based IMS, in which the matrix crystallization process is eliminated. In this chapter, a practical protocol for MALDI-IMS of lipids is outlined. In addition, as an attractive alternative to MALDI-based IMS, we also present nanoparticle-based IMS that improves spatial resolution.