In the mammalian DNA damage response, ADP-ribosylation signalling is of crucial importance to mark sites of DNA damage as well as recruit and regulate repairs factors. Specifically, the PARP1:HPF1 complex recognises damaged DNA and catalyses the formation of serine-linked ADP-ribosylation marks (mono-Ser-ADPr), which are extended into ADP-ribose polymers (poly-Ser-ADPr) by PARP1 alone. Poly-Ser-ADPr is reversed by PARG, while the terminal mono-Ser-ADPr is removed by ARH3. Despite its significance and apparent evolutionary conservation, little is known about ADP-ribosylation signalling in non-mammalianAnimalia. The presence of HPF1, but absence of ARH3, in some insect genomes, includingDrosophilaspecies, raises questions regarding the existence and reversal of serine-ADP-ribosylation in these species. Here we show by quantitative proteomics that Ser-ADPr is the major form of ADP-ribosylation in the DNA damage response ofDrosophila melanogasterand is dependent on thedParp1:dHpf1 complex. Moreover, our structural and biochemical investigations uncover the mechanism of mono-Ser-ADPr removal byDrosophilaParg. Collectively, our data reveal PARP:HPF1-mediated Ser-ADPr as a defining feature of the DDR inAnimalia. The striking conservation within this kingdom suggests that organisms that carry only a core set of ADP-ribosyl metabolising enzymes, such asDrosophila, are valuable model organisms to study the physiological role of Ser-ADPr signalling.