Hydrodynamic drag forces generated by liquid flow above a supported lipid bilayer (SLB) can be used to induce lateral movement of molecules protruding from the SLB. Since the velocity of the individual molecules depends on their size and coupling to the lipid bilayer, these forces can also be used to enrich and separate different types of membrane-bound molecules. To improve and better quantify hydrodynamic-based molecular separation in SLBs, we formed the SLB on the floor of a microfluidic channel which was patterned with gold barriers that confined the lipid bilayer to a 100 mm wide strip in the center of a 300 mm wide microfluidic channel. This forces the SLB into a region of the channel where the spatial variation of the hydrodynamic forces is close to zero while at the same time preventing the SLB from creeping up on the PDMS sides of the channel, thus reducing the loss of material. We here use this approach to investigate the accumulation of (i) fluorescently labeled lipids and (ii) the protein complex cholera toxin B (CTB) and to compare how the accumulation and separation differ when having an infinite reservoir or only a spatially limited band of studied molecules in the SLB. In addition, we show how the method can be used for complete separation of different polyvalently bound fractions of CTB.