Solid supported lipid bilayers (SLBs) serve as an excellent platform for biophysical studies. However, the formation of highly negatively charged SLBs on negatively charged surfaces remains a challenge due to electrostatic repulsion. Here, we study the effects of ionic strength and osmotic stress on the formation of highly negatively charged SLBs on the silica surface. We used quartz crystal microbalance-dissipation to study the adsorption and rupture of highly negatively charged small unilamellar vesicles on the silica surface in different concentrations of NaCl and under different osmotic stresses. It was demonstrated that an increase in the ionic strength of the solution enhances SLB formation. Both hypertonic and moderate hypotonic osmotic stress can promote the formation of SLBs. However, the SLB cannot be formed under high hypotonic osmotic stress. Importantly, osmotic stress alone without a change in ionic strength is insufficient to promote SLB formation. Moreover, the topographical images obtained by atomic force microscopy showed that complete bilayers were formed under hypertonic osmotic stress and high ionic strength, whereas defects were noticed in the bilayers formed under hypotonic osmotic stress. Furthermore, the fluidity of the lipid bilayers was studied by fluorescence recovery after photobleaching. A higher membrane fluidity was observed for the complete lipid bilayers compared to that of the lipid bilayers with defects. Our findings further the understanding of how ionic strength and osmotic stress affect the formation of highly negatively charged SLBs on negatively charged surfaces, providing insights for preparing model biological membranes.