Biosensor technology was employed to study the specific interactions of different lipopolysaccharide (LPS)-binding proteins and peptides with LPS, using an LPS-coated surface. Two methods to immobilize biotinylated LPS to streptavidin-coated sensor chips (SA-chips) were evaluated. Biotinylated LPS in PBS or biotinylated LPS, pretreated with EDTA and sodium-desoxycholate, were injected across an SA-chip, resulting in a 'high-' and 'low- mass' LPS chip, respectively. While the 'high mass' LPS chip appeared to be unstable, the 'low mass' LPS chip resulted in reproducible binding curves for bactericidal/permeability-increasing protein (rBPI21) with a binding affinity corresponding to the literature (Kd: 3.75 nM). New Kd values were obtained for serum amyloid P component (SAP, Kd: 3.9 nM), a recently discovered new LPS-binding protein, and cationic protein 18 (CAP18, Kd: 0.58 nM). Moreover, binding affinities of bioactive BPI- and SAP-derived peptides could be determined. This study shows for the first time the applicability of biosensor technology to study interactions of proteins and peptides with LPS, using an LPS-coated sensor chip.