Microtubules form a major cytoskeleton and exhibit dynamic instability through the repetitive polymerization/depolymerization of tubulin dimers. Although microtubule stability should be precisely controlled to maintain various cellular functions, it has been difficult to assess its status in vivo. Here, we propose a tubulin fractionation method reflecting the stability of microtubules in mouse tissues. Analyses of tubulin fractionated by two-step of ultracentrifugation demonstrated three distinct pools of tubulin, that appeared to be stable microtubule, labile microtubule, and free tubulin. Using this method, we were able to show the specific binding of different microtubule-associated proteins onto each pool of microtubules. Also, there were clear differences in the population of stable microtubule among tissues depending on the proliferative capacity of the constituent cells. These findings indicate that this method is useful for broad analysis of microtubule stability in physiological and pathological conditions.