In this chapter, we demonstrate the technique of TLC-imaging mass spectrometry consisting of thin-layer chromatography (TLC), the TLC-blot method, and imaging mass spectrometry. By the use of this method, we could separate, purify, detect, and identify very faint lipids at the same time. We could detect a lipid at a low-picomole level; this method shows higher sensitivity than the conventional staining method. This method is also suitable for molecular species differential analysis by constructing the ion image of each molecular species with fine resolution (micrometer order). Furthermore, this system is capable of quantitative analysis by the use of TLC. It needs no special equipment or technique, and thus it is easy for everyone to begin using.