To investigate the regulation of protein phosphorylation during the DNA damage response and the role of ATR kinase activity, we performed a comparative phosphoproteomic analysis of HeLa cells treated with etoposide alone or in combination with the ATR inhibitor VE821. Etoposide is a topoisomerase II inhibitor that induces DNA double-strand breaks, activating the DNA damage signaling cascade. We focused particularly on phosphorylation events associated with Treacle (TCOF1), a nucleolar protein implicated in the regulation of rDNA transcription and nucleolar DNA damage response. Quantitative mass spectrometry-based phosphoproteomics was used to identify and quantify global phosphorylation changes under both treatment conditions. This dataset enables the identification of ATR-dependent phosphorylation sites and may provide insights into the molecular mechanisms underlying nucleolar stress responses and the coordination of DNA repair processes.