A new miniature streaming potential apparatus (SPA) was developed to determine the streaming potentials (Ψstr) and zeta potentials (ζ) of substrates under different ionic conditions while simultaneously visualizing the state of the surfaces, such as the adsorption of surfactants or polymers, using fluorescence microscopy and/or fluorescence recovery after photobleaching (FRAP). Experimental results with different surfaces show that the new SPA provides streaming potential values (hence zeta potentials) that agree with results obtained using traditional electrokinetic analyzers. Using the new SPA, the formation of supported lipid bilayers (SLBs) on glass from fluorescently labeled, unilamellar (100 nm diameter), charge neutral dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles was studied in aqueous electrolyte solutions at different lipid concentrations. Simultaneous zeta potential measurements and fluorescence imaging for measuring diffusion coefficients by confocal microscopy enabled us to precisely monitor the changes in the surface charge as well as in the surface morphology during SLB formation from vesicles. For a fixed incubation time of 5 min, both results revealed that the adsorption of intact vesicles and/or discrete bilayer patches were observed below a threshold concentration, above which the formation of continuous SLBs occurred leading to an estimate for the ζ-potential and for the diffusion coefficient of −9.1 ± 1.6 mV and (1.1 ± 0.02) × 10−12 m2/s, respectively.