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Static secondary ion mass spectrometry (SSIMS) is a capable of providing detailed atomic and molecular characterization of the surface chemistry of biological and biomedical materials. The technique is particularly suited to the detection and imaging of small molecules such as membrane lipids, metabolites, and drugs. A limit of detection in the ppm range and spatial resolution <1 μm can be obtained. Recent progress in instrumental developments, notably cluster ion beams, and the application of multivariate data analysis protocols, promise further advances. This chapter presents a brief overview of the theory and instrumentation of static secondary ion mass spectrometry followed by examples of a range of biological and biomedical applications. Because of the ultrahigh vacuum requirements and extreme surface sensitivity of the technique, appropriate sample preparation and handling is essential. These protocols, and the analysis methodology required to ensure high-quality, reliable data are described.