Polo-like kinase 1 (Plk1) is an important protein kinase for checkpoint recovery and adaptation in response to DNA damage and replication stress. However, although Plk1 is present in S phase, little is known about its localization and function during unperturbed DNA replication. Here used recombinant XRif1 C-terminal domain and recombinant XPlk1 in an in vitro phosphorylation assay followed by LC/MS/MS to identify which residues are phosphorylated by Plk1 on Rif1 CTD.