The resulting compositional variability is assessed either via external calibration curves which determine absolute concentrations of isotopically labeled lipids in an SLB or by examining the relative concentrations of two isotopically labeled lipids. This second method avoids relying on the accuracy of external standards and is not subject to potential contamination of the NanoSIMS substrate. Both methods demonstrate that patches formed from individual GUVs show considerable composition variation, and that GUVs prepared via electroformation are less variable than GUVs formed via gentle hydration. Additionally, quantification of the average concentration of cholesterol in GUVs formed via gentle hydration and electroformation suggest that GUVs formed by electroformation have significantly less cholesterol on average than GUVs formed via gentle hydration.