The Protein Lipid Overlay (PLO) assay enables the identification of the lipid ligands with which lipid binding proteins interact. This assay also provides qualitative information on the relative affinity with which a protein binds to a lipid. In the PLO assay, serial dilutions of different lipids are spotted onto a nitrocellulose membrane to which they attach. These membranes are then incubated with a lipid binding protein possessing an epitope tag. The membranes are washed and the protein, still bound to the membrane by virtue of its interaction with lipid(s), is detected by immunoblotting with an antibody recognizing the epitope tag. This procedure requires only a few micrograms of protein and is quicker and cheaper to perform than other methods that have been developed to assess protein-lipid interactions. The reagents required for the PLO assay are readily available from commercial sources and the assay can be performed in any laboratory, even by those with no prior expertise in this area.