ADP-ribosylation is a post-translational modification involved in DNA damage response (DDR). In higher organisms it is synthesised by PARP 1–3, DNA strand break sensors. Recent advances have identified serine residues as the most common targets for ADP-ribosylation during DDR. To ADP-ribosylate serine, PARPs require an accessory factor, HPF1 which completes the catalytic domain. Through ADP-ribosylation, PARPs recruit a variety of factors to the break site and control their activities. However, the timely removal of ADP-ribosylation is also key for genome stability and is mostly performed by two hydrolases: PARG and ARH3. Here, we describe the key writers, readers and erasers of ADP-ribosylation and their contribution to the mounting of the DDR. We also discuss the use of PARP inhibitors in cancer therapy and the ways to tackle PARPi treatment resistance.