Matrix-assisted laser desorption/ionization (MALDI)-imaging mass spectrometry (IMS; also referred to as mass spectrometry imaging, MSI) enables the visualization of the distribution of variable biomolecules that have large and varied structures in tissue sections. However, because of such general versatility, the optimization of sample preparation procedure according to each analyte with distinct chemical and physical properties is an important task for IMS, similar to that in traditional MALDI-MS analyses. In this chapter, we briefly introduce the sample preparation strategies of current IMS. We describe the two major strategies for imaging of proteins/peptides and small molecules, such as lipids and drugs. We shall also introduce the representative application example.