© 2016 Elsevier Inc. Septins are highly conserved and essential eukaryotic cytoskeletal proteins that interact with the inner plasma membrane. They are involved in essential functions requiring cell membrane remodeling and compartmentalization, such as cell division and dendrite morphogenesis, and have been implicated in numerous diseases. Depending on the organisms and on the type of tissue, a specific set of septins genes are expressed, ranging from 2 to 13. Septins self-assemble into linear, symmetric rods that can further organize into linear filaments several microns in length. Only a subset of human septins has been described at high resolution by X-ray crystallography (Sirajuddin et al., 2007). Electron microscopy (EM) has proven to be a method of choice for analyzing the molecular organization of septins. It is possible to localize each septin subunit within the rod complex using genetic tags, such as maltose-binding protein or green fluorescent protein, to generate a visible label of a specific septin subunit in EM images that are processed using single-particle EM methodology. In this chapter we present, in detail, the methods that we have used to analyze the molecular organization of budding yeast septins (Bertin et al., 2008). These methods include purification of septin complexes, sample preparation for EM, and image processing procedures. Such methods can be generalized to analyze the organization of septins from any organism.