Developments in chemical protein synthesis have enabled the generation of tailor-made proteins including incorporation of many types of modifications into proteins, enhancing our ability to control site-specificity of protein posttranslational modifications (PTMs), modify protein backbones and introduce photocrosslinking probes. For PDZ (postsynaptic density protein, disks large, zonula occludens) protein domains, expressed protein ligation (EPL) has been employed to introduce analogs of cognate amino acids, amide-to-ester bond mutations, and phosphorylations in the study of PDZ domain-mediated protein-protein interactions (PPIs). Here, we present protocols for EPL of PDZ domains focusing on phosphorylation and amide-to-ester modifications in the PDZ domain proteins.