The binding of an amphipathic ␣-helical peptide to small unilamellar lipid vesicles has been examined using chemical derivitization and mass spectrometry. The peptide is derived from the sequence of human apolipoprotein C-II (apoC-II), the protein activator of lipoprotein lipase (LpL). ApoC-II 19 -39 forms approximately 60% ␣-helix upon binding to model egg yolk phosphatidylcholine small unilamellar vesicles. Measurement of the affinity of the peptide for lipid by spectrophotometric methods is complicated by the contribution of scattered light to optical signals. Instead, we characterize the binding event using the differential labeling of lysine residues by the lipidand aqueous-phase cross-linkers, disuccinimidyl suberate (DSS) and bis(sulfosuccinimidyl) suberate (BS 3 ), respectively. In aqueous solution, the three lysine residues of the peptide are accessible to both cross-linkers. In the presence of lipid, the C-terminal lysine residue becomes inaccessible to the lipid-phase crosslinker DSS, but remains accessible to the aqueousphase cross-linker, BS 3 . We use mass spectrometry to characterize this binding event and to derive a dissociation constant for the interaction (K d ؍ 5 M). We also provide evidence for the formation of dimeric cross-linked peptide when high densities of peptide are bound to the lipid surface.