Poly(ADP-ribose) (pADPr) is a heterogenic molecule synthesised from NAD by poly(ADP-ribose) polymerases (PARPs). Many cellular functions from genome integrity surveillance, cell cycle progression and DNA repair to apoptosis are affected by pADPr through its network of associated proteins. Using quantitative proteomics, we established a temporal map of pADPr-associated complexes upon genotoxic stress. Results suggested a strong pADPr association to many proteins involved in stress granule formation, notably the ras-GAP SH3-binding protein G3BP, as well as in the later phases of alkylation-stress-induced responses. Further investigation with dynamic imaging clearly demonstrated a pADPr-dependent initiation of stress granule assembly originating from the nucleus. The co-transfection of G3BP with poly(ADP-ribose) glycohydrolase (PARG) indicates that pADPr is involved in modulating the nuclear translocation of G3BP. Moreover, a peptide pADPr blot assay of G3BP revealed that pADPr binds to the glycine-arginine-rich domain of G3BP. Thereafter, we established a comprehensive G3BP interactome in the presence of pADPr. Our findings establish a novel function for pADPr in the formation of G3BP-induced stress granules upon genotoxic stress.