Mass spectrometry imaging (MSI), derived from the study of material surfaces by secondary ion mass spectrometry, traces its origins to the emergence of the Laser Microprobe Mass Analyzer (LAMMA) concept in the mid-1990s. Subsequently, the LAMMA concept evolved into Matrix-Assisted Laser Desorption Ionization (MALDI), a novel molecular visualization technique first reported in the United States and Europe. At its initial introduction, the focus was primarily on protein imaging due to the prominence of protein ionization, which was later honored with the Nobel Prize in Chemistry in 2002. Today, however, the dominant trend is the imaging of small molecules such as metabolites, lipids, and pharmaceuticals in living organisms. In this review, I provide an overview of the various MSI methods, explain the underlying principles driving the advancement of mass microscope which pioneered MSI research in Japan, and present illustrative examples of the integration of MSI technique into clinical trials. In addition, I will describe the enzyme histochemistry method using MSI that my research group is currently pursuing in addition to hair analysis conducted by my venture company.