Matrix Assisted Lazar Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS) is the novel method to visualize molecules in tissue specimens without any labeling. However, if we obtained the localization information of trace amount of molecular species, the insufficient detection sensitivity will become one of the biggest problems. Since there are no separation or purification process in MALDI-IMS, ionization suppression caused by contaminants derived from living tissue samples will significantly make an impact on detection sensitivity. Additionally, molecules that have the difficult-to-ionize characteristics will become problems. Therefore, we developed the combinations of on-tissue derivatization, matrix additives and MS/MS analysis to reduce the ionization suppression and to improve the ionization ratio of difficult-to-ionize molecules such as prostaglandins.