The adsorption and spreading of lipid vesicles on solid supports has become a popular way to create supported lipid bilayers (SLBs), but little attention has been paid to the possible redistribution of lipid material between the two leaflets of an SLB. We use the technique of quartz crystal microbalance with dissipation monitoring (QCM-D) to follow the adsorption of prothrombin on SLBs formed from sonicated unilamellar vesicles containing mixtures of dioleoylphosphatidylcholine (DOPC) and dioleoylphospatidylserine (DOPS). The specific interaction of prothrombin with negatively charged lipids is quantified and serves as a reporter of the content of accessible DOPS in SLBs. We compare results obtained on silica and mica and find that the underlying support can induce substantial redistribution of lipid material between the two leaflets. In particular, SLBs formed on mica showed a substantially depleted amount of accessible DOPS in the presence of calcium. The mechanisms that lead to the lipid redistribution process are discussed.