A miniaturized streaming potential apparatus (SPA) was newly developed in order to measure surface potentials under different solution conditions while simultaneously visualizing the state of the surfaces such as the adsorption of vesicles or bilayers using fluorescence microscopy or fluorescence recovery after photobleaching (FRAP) technique. Two different sets of substrates (such as Teflon against borosilicate and Teflon) against Teflon under different buffer solution concentrations were used to validate our new design of the SPA. Our results obtained from the SPA show good agreements in both cases with possible predictions based on a modified Helmholtz-Smoluchowski (H-S) equation. Certain amount of DMPC (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine) vesicle solutions were introduced into the SPA chamber to investigate how the zeta (ζ)-potential would be changed as the formation of DMPC bilayers on a borosilicate surface. Simultaneous ζ-potential measurements and fluorescence imaging and diffusion coefficients by confocal microscopy using the new SPA allow the monitoring of bilayer formation from vesicles as a function of lipid concentrations.