The development of mass spectrometry has recently entered a new phase. The previous limitation of mass analyses of biomolecules confined the imagination of researchers to a single location from which samples were gathered; however, an innovation in mass spectrometry has now enabled the provision of additional two-dimensional (2D) axes of recognition on tissue sections. The innovatory technology — imaging mass spectrometry — can visualize the distribution of molecules. It has attracted a great deal of attention in the analyses of drug effects, in screening of drugs, and in support for medical diagnoses. In this chapter, we describe imaging mass spectrometry as an historical innovation and the significance of publishing this protocol book. I strongly believe that this book provides a critical basis for future studies on imaging mass spectrometry.