Lysophosphatidic acid (LPA) is a lipid mediator which regulates various biological processes, including cell proliferation, platelet aggregation, and cancer metastasis. Autotaxin (ATX) is a lysophospholipase D (lyso-PLD)-type enzyme that produces LPA in plasma. In contrast, glycerophosphodiesterase GDE4 and GDE7 (GDEs) are intracellular lyso-PLDs producing LPA. ATX and GDEs may play unique roles, which need to be validated with their potent and selective inhibitors. Although fluorescent substrates are useful for high-throughput screening of enzyme inhibitors, no fluorescent substrates for these GDEs have been reported. Here, we examined whether a fluorescent ATX substrate FS-3 could be applied to such assays. The membrane fractions of GDE4- and GDE7-overexpressing cells hydrolyzed FS-3 in the presence of Mg²⁺ and Ca²⁺, respectively. Using these assay systems, we found that several known ATX inhibitors as well as natural substrates lysophosphatidylcholine and lysophosphatidylethanolamine inhibited GDEs. Furthermore, FS-3 was hydrolyzed by the membrane fraction of LNCaP and MCF-7 cells that endogenously express GDE4 and GDE7, respectively. Finally, our assay system could selectively measure GDEs activities in a mixture of the membrane fractions of GDEs-overexpressing cells. These findings allow high-throughput assays of GDEs and lead to the development of selective inhibitors as well as a better understanding of the biological roles of these enzymes.