There are many different techniques available to investigate component distribution within cellular and model membranes. However, analysis of membrane composition is still challenging. We have developed an approach to explore the use of secondary ion mass spectroscopy, which is performed with a NanoSIMS 50 (Cameca), to elucidate the distribution of components in supported lipid bilayers. Lateral resolution of 50 nm and high sensitivity at high mass resolution can be achieved with this instrument. A focused cesium ion beam is rastered across the sample, extensively fragmenting the supported membrane. Up to five different secondary ions are simultaneously detected. By incorporating distinct stable isotopes into each membrane component, the secondary ion signals characteristic of each labeled species are used to image and quantify the membrane composition within small regions of a supported lipid membrane. Here we use this approach to investigate the distribution of cholesterol within phase separated supported lipid bilayers. Ultimately, this approach may be applied to investigate the existence of cholesterol-enriched domains in cellular membranes.