Microtubules are essential cellular structures in plant cells. They are polymerized from tubulin dimers and are regulated by microtubule-associated proteins (MAPs). Here, we describe a protocol for purifying tubulin dimers and MAPs from plant cells. The protocol involves preparing vacuole-free mini-protoplasts, a high quality cytoplasmic extract, cycles of microtubule polymerization and depolymerization to increase tubulin and MAP concentration, separation of tubulin and MAPs by column chromatography. We also present tubulin purification methods for biochemical assays.