Phosphoinositides derive from the phospholipid, phosphatidylinositol (PtdIns), by phosphorylation of the inositol ring in the lipid head group. The determination of phosphoinositide species is a particular challenge, because the structurally similar inositolphosphate-head groups must be analyzed as well as the lipid-associated fatty acids. The method presented in this chapter consists of two steps: First phosphoinositides are separated by thin-layer chromatography (TLC) according to their characteristic head groups and the individual lipids are isolated. Second, the fatty acids associated with each isolated lipid are analyzed using a gas-chromatograph (GC). The combination of these two classical methods for lipid analysis, TLC and GC, provides a cost-efficient and reliable alternative to lipidomics approaches requiring more extensive instrumentation.