Microtubules are highly dynamic structures which play a central role in many cellular processes such as cell division, intracellular transport, and migration. Their dynamics is tightly regulated by stabilizing and destabilizing microtubule-associated proteins (MAPs), such as tau and stathmin. Many approaches have been developed to study interactions between tubulin and MAPs. However, isothermal titration calorime-try (ITC) is the only direct thermodynamic method that enables a full thermodynamic characterization of the interaction after a single titration experiment. We provide here the protocols to apply ITC to tubulin interaction with either stathmin or tau, which will help to avoid the common pitfalls in this very powerful and sensitive method.