Updated publication reference for PubMed record(s): 30157440. Here we report systems-wide identification of serine ADP-ribosylation sites and quantification of their cellular changes in human cells upon oxidative stress. High-resolution mass spectrometry and unrestricted data processing confirmed that serine residues are the major target of ADP-ribosylation in HeLa cells. Proteome-wide analyses identified 3,090 serine ADP-ribosylation sites, with 97 percent of acceptor sites modulated more than 2-fold upon oxidative stress, while treatment with PARP inhibitor Olaparib abrogates induction (part 1, ADP-ribosylation data, PXD009208). Consistent with the nuclear expression of ARTD1/PARP1 and ARTD2/PARP2, serine ADP-ribosylation prominently occurred on nuclear proteins. Structural-predictive analyses revealed that serine ADP-ribosylation preferentially resides in disordered regions, and identified serine ADP-ribosylation sites to significantly overlap with known phosphorylation sites. Large-scale phosphoproteomics analysis supported these observations (part 2, phosphorylation data), hereby providing first evidence for site-specific crosstalk between serine ADP-ribosylation and phosphorylation. Collectively, we demonstrate that serine ADP-ribosylation is a widespread modification and a major nuclear responder to oxidative stress, and that its regulatory scope is comparable to other posttranslational modifications.