Post-translational modifications (PTMs) alter the molecular structure, localization, function, and stability of proteins, and play central roles in a wide range of biological processes and diseases. Nowadays, various PTMs, such as phosphorylation, acetylation, and glycosylation, can be efficiently discovered by proteomic studies. However, it is still challenging and laborious to address the function of any modification by biochemical and genetic approaches. We apply genetic code expansion strategy for introducing post-translational modifications. With this technology, authentic PTMs can be co-translationally incorporated into protein of interest at desired positions without knowing specific modifying writer enzymes, allowing efficiently study the effect of the modifications on protein.