Septins are polymerizing eukaryotic proteins that play conserved roles in cell cortex organization and are essential in many cell types. How septin dynamics and protein-protein interactions determine their function at the plasma membrane remains a mystery. Here, we present a method for recapitulating septin polymerization and lipid interaction utilizing supported lipid bilayers to mimic the eukaryotic plasma membrane. Septins on supported lipid bilayers can be visualized with single-molecule sensitivity using total internal reflective fluorescence microscopy. Microscopy-based in vitro assays have revolutionized our understanding of actin, microtubules, and bacterial cytoskeletal systems, and will likely immediately advance our understanding of the septin proteins. As such, we hope that this technique will be adopted and widely utilized by those interested in uncovering septin properties and functions of septin interacting proteins.