Despite their widespread use, there is some evidence pointing to potential issues with GUVs as model systems. In particular, prior work has shown that electroformed GUVs composed of a ternary mixture exhibit significant variation in areas occupied by an Ld partitioning fluorescent dye. Other work has shown that individual GUVs produced from the same lipid film show significant variations in their phase behavior . Although work has been done to compare GUV formation methods in terms of their resulting unilamellarity, capacitance, shear viscosity and number of defects visible by fluorescence microscopy , compositional variability has yet to be directly examined. Quantifying this variability is critical as lipid composition is the primary variable in all GUV-based measurements. Here, we examine the compositional variability between individual GUVs using stable isotope labeling and secondary ion mass spectrometry (SIMS).