Poly(ADP-ribose) (pADPr) is heterogenic molecule synthesized from NAD by poly(ADP-ribose) polymerases (PARPs). Multiple cellular functions are affected by pADPr through its network of associated proteins ranging from genome integrity surveillance, cell cycle progression, DNA repair to apoptosis. Using quantitative proteomics, we established a temporal map of pADPr-associated complexes upon genotoxic stress. Results suggested a strong pADPr-association of multiple proteins involved in stress granule formation, notably G3BP, in latter phases of alkylation-stress-induced cells. 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 shuttling 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 presence of pADPr. Our findings establish a novel function for pADPr in the formation of G3BP-induced stress granules upon genotoxic stress.