Interactions of lipids with proteins are essential events in the framework of biological membranes. Assessment of the affinity and specificity of protein-lipid binding can give useful information to elucidate cell membrane functions. Surface Plasmon Resonance (SPR) is a powerful technology to study macromolecular interactions, allowing direct and rapid determination of association and dissociation rates using small amounts of samples. An extensive range of binding analyses can be performed by SPR such as protein–protein, protein–membrane (lipids), protein–carbohydrate, protein–nucleic acid and even protein-small molecules. This protocol describes the binding of an antimicrobial protein (used as ligand) to a lipopolysaccharide (LPS) (used as analyte) after immobilization onto a CM sensor chip by amine coupling.