Matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) is an emerging label-free method for mapping the distribution of diverse molecular species in tissue sections. Despite recent progress in MALDI-MSI analyses of lipids, it is still difficult to visualize minor bioactive lipids including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). Here, we have developed a novel on-tissue derivatization method using Phos-tag, a zinc complex that specifically binds to a phosphate monoester group. MALDI-MSI with Phos-tag derivatization made it possible to image LPA and S1P in the murine brain. Furthermore, we were able to visualize other low-abundance lipids containing phosphate monoester, such as phosphatidic acid and ceramide-1-phosphate. Compared with conventional MALDI-MS, this derivatization produced LPA images with high spatial accuracy discriminating LPA artificially produced during MALDI-MS analysis. In mice with deficiencies in enzymes that degrade LPA and S1P, we observed marked S1P and/or LPA accumulation in specific regions of the brain. Thus, the present study provides a simple and optimal way to reveal the spatial localization of potent bioactive lipid phosphates such as LPA and S1P in tissues.