Mass spectrometry (MS) is an excellent technology for molecular imaging because of its high data dimensionality. MS can monitor thousands of individual molecular data channels measured as mass-to-charge (m/z). We describe the use of matrix-assisted laser desorption/ionization (MALDI) MS for the image analysis of proteins, peptides, lipids, drugs, and metabolites ill tissues. We discuss the basic instrumentation and sample preparation methods needed to produce high-resolution images and high image reproducibility Matrix-addition protocols are briefly discussed;along with normal operating procedures, mid selected biological and medical applications of MALDI imaging MS are described. We give examples of both two- and three-dimensional imaging, including normal mouse embryo implantation, sperm maturation ill mouse epididymis, protein distributions in brain sections, protein alterations is :1 result of drug administration,and protein changes in brain due to neurodegeneration and tumor formation. Advantages of this technology and future challenges for its improvement are discussed.