Classical approaches for probing protein phosphorylation events rely on phosphomimicking amino acids or enzymatic phosphorylation of proteins. In many cases, phosphomimicking amino acids inadequately imitate actual protein phosphorylation, whereas the latter method suffers from an inability to control site specificity and stoichiometry. To circumvent these shortcomings, chemical biological approaches have been developed to enable introduction of phosphorylated amino acids into proteins in a reliable and controlled way. Here, we describe methods to make semisynthetic, phosphorylated PDZ domains, covering expressed protein ligation (EPL) strategies involving modifications within the N-terminal or C-terminal regions. We also enclose protocols for the biophysical characterization of the semisynthetic phosphorylated PDZ domains to establish whether the introduced phosphorylation affects protein structure, stability, and function.