Giant unilamellar vesicle (GUV) formation on indium tin oxide (ITO) electrodes in saline solution and from charged lipids has proven to be difficult in the past. Yet the best cell membrane models contain charged lipids and require physiological conditions. We present a way to overcome this problem by using plasma cleaned ITO electrodes. GUVs from zwitterionic lipids, lipid mixtures and even pure charged lipids could be electroformed under physiological conditions and even higher concentrations of NaCl. The hydrophilic ITO surface may facilitate the hydration of the solid lipid film and the formation of lipid bilayers that subsequently bend and form vesicles. The formation of GUVs in saline solution is influenced by different parameters. The influences of the amplitude and frequency of the used AC field, the NaCl concentration, and the temperature were investigated. Finite element analysis simulating the effect of the electric field on GUV formation in saline solution could well explain the experimental results. Frequencies in the kHz-range favored for GUVs formation in saline solution, as they suppress the formation of electric double layer, while higher frequencies could again impair the effect of electric field and impede GUV formation. The diameters of the GUVs increased gradually with NaCl concentration from 0mM to 200mM and subsequently decreased from 200mM to 2M. High yields of GUVs were also formed in PBS solution and cell culture medium, which indicates this method is a promising way to prepare GUVs on a large scale in physiological relevant conditions.