The DNA damage response revolves around transmission of information via post-translational modifications, including reversible protein ADP-ribosylation. Here, we applied a massspectrometry-based Af1521 enrichment technology for the identification and quantification of ADPribosylation sites as a function of various DNA damage stimuli and time. In total, we detected 1681 ADP-ribosylation sites residing on 716 proteins in U2OS cells and determined their temporal dynamics after exposure to the genotoxins H2O2 and MMS. Intriguingly, we observed a widespread but low-abundance serine ADP-ribosylation response at the earliest time point, with later time points centered on increased modification of the same sites. This suggests that early serine ADPribosylation events may serve as a platform for an integrated signal response. While treatment with H2O2 and MMS induced homogenous ADP-ribosylation responses, we observed temporal differences in the ADP-ribosylation site abundances. Exposure to MMS-induced alkylating stress induced the strongest ADP-ribosylome response after 30 min, prominently modifying proteins involved in RNA processing, whereas in response to H2O2-induced oxidative stress ADP-ribosylation peaked after 60 min, mainly modifying proteins involved in DNA damage pathways. Collectively, the dynamic ADP-ribosylome presented here provides a valuable insight into the temporal cellular regulation of ADP-ribosylation in response to DNA damage.