A novel approach for multiplexed biomolecule detection at surfaces, involving specific binding of liposomes and subsequent analysis by time-of-flight secondary ion mass spectrometry (ToF-SIMS), was evaluated with respect to its capability for quantitative analysis of biomolecule surface concentrations. The specific binding of liposomes to a poly(L-lysine)-g-poly (ethylene glycol) (PLL-g-PEG) surface, using the biotin-avidin coupling chemistry, was characterized by quartz crystal microbalance with dissipation monitoring (QCM-D), fluorescence microscopy and ToF-SIMS. The ToF-SIMS results showed a linearly increasing signal from the liposomes up to a saturation coverage corresponding to a full liposome layer on the surface, in close agreement with fluorescence microscopy analysis of the same samples, strongly supporting the potential of the liposome-based approach for quantitative biomolecule detection. However, the multiplexing capability and issues on nonspecific binding need further studies. Furthermore, an improved method for the preparation of lipid bilayer samples for ToF-SIMS analysis is presented, demonstrating the imaging of individual 210-nm diameter liposomes adsorbed on a SiO2 surface.