Microtubules, dynamic polymers assembled from α, β-tubulin dimers, contribute to myriad cellular processes. This is largely attributed to microtubule-associated proteins (MAPs). How MAPs selectively bind microtubules to carry out various functions is not known. The “Tubulin Code” theory proposes that posttranslational modifications (PTMs) of microtubules serve as signs that can be read by specific MAPs, thereby conferring specific functional properties to the microtubules. In support of this hypothesis, “reader” MAPs have been identified for various tubulin PTMs, but, until recently, no systematic screening had been performed to identify readers in an unbiased manner. We addressed this by developing a reader identification pipeline that uses quantitative mass spectrometry to interrogate the microtubule proteome of cells programmed to express specific PTMs. This pipeline can be used to identify readers for any tubulin PTM from various cell types as long as the writer enzymes are known. We also provide an alternative, complementary approach to obtain modified microtubules using a generic writer enzyme in vitro.