Microtubules play essential roles in numerous cellular processes. All microtubules are built from the protein tubulin, yet individual microtubules can differ spatially and temporally due to their tubulin isotype composition and post-translational modifications (PTMs). The tubulin code hypothesis posits that these differences can regulate microtubule function. However, investigating the properties of specific tubulin PTMs in vitro has been challenging because most reconstitution assays rely on tubulin purified from brain tissue that contains highly heterogeneous and modified microtubules. In this study, we present an optimized method for the purification of milligram quantities of unmodified tubulin from large-scale cultures of HeLa S3 cells. We also describe steps for efficient chemical labeling of tubulin and the generation of controlled tubulin PTMs. These tubulins can be used in microscopy or biochemistry-based experiments to investigate how the tubulin code influences microtubule properties and functions. Overall, our method is easily adaptable, highly reproducible, and broadly accessible to labs with general equipment.