Microtubules are dynamic polymers where tubulin exchanges not only at the ends but also all along the microtubule shaft. In vitro reconstitutions are a vital approach to study microtubule tip dynamics, while direct observation of shaft dynamics is challenging. Here, we describe a dual-color in vitro assay to visualize microtubule shaft dynamics using purified, labeled bovine brain tubulin. With this assay, we can quantitatively address how proteins or small molecules impact the dynamics at the microtubule shaft. For complete details on the use and execution of this protocol, please refer to Andreu-Carbó et al. (2022).