Microtubules are cytoskeletal structures built of alpha- and beta-tubulins. Although tubulins are highly conserved throughout evolution, microtubules can be adapted to a range of different functions. A powerful mechanism that could regulate the functional specialization of microtubules is the posttranslational modification of tubulin molecules. Two tubulin modifications, polyglutamylation and polyglycylation, generate amino acid side chains of different length on tubulin. These modifications are thought to regulate interactions between microtubules and their associated proteins; however, detailed studies of this potential mechanism have not been performed. The investigation of the potential regulatory role of polyglutamylation requires in vitro tools to visualize the molecular events that could be affected by this modification. Classically, in vitro work with microtubules is performed with tubulin from brain tissue; however, this tubulin is highly posttranslationally modified. Here, we describe a method for the purification of tubulin carrying controlled levels of polyglutamylation, which can be used in basic in vitro assays.