INTRODUCTIONFluorescently tagged lipid-binding domains have become a popular tool to image lipids that are involved in intracellular signaling processes. The readout usually involves the translocation of the lipid-binding domain from the cytosol or nucleosol to the membrane of interest, or vice versa. Unfortunately, this method seems to work predominantly for lipids in the plasma membrane, whereas lipids such as phosphatidylinositol 4,5-bisphosphate (PIP₂) are not recognized in the membranes of the endoplasmic reticulum or the Golgi. Very recently, we developed an alternative way of localizing a lipid of interest by fluorescent labeling of minimally modified lipid derivatives using a single specific chemical reaction. This protocol describes how to directly label lipids in fixed cells for lipid location analyses.