Microtubules are rigid and highly dynamic cellular polymers essential for intracellular transport, cell division and differentiation. Their stability is tightly regulated by a vast array of cellular factors. In vitro microtubule assays have proven to be powerful tools for deciphering the mechanism of microtubule dynamics regulators such as molecular motors and microtubule associated proteins. In this chapter we focus on microtubule severing enzymes that use the energy of ATP hydrolysis to introduce internal breaks in the microtubule lattice. We present a detailed protocol for a light microscopy based in vitro microtubule severing assay that was instrumental in the identification and characterization of these enzymes.