The histone H2AX has been established as a reliable biomarker of DNA damage, becoming phosphorylated rapidly following damage in discrete foci which can be correlated with DNA DSB number. In the present study, the phosphorylation of H2AX was used as a marker of DNA DSB damage to compare and contrast the damage induced by ionizing radiation, the topo II poison, etoposide, and the topo II catalytic inhibitor, ICRF-193. To examine the DNA damage numbers at time-points in the 24 hours following exposure to these damaging agents. Topo lIP null cells were used to investigate the contribution of topo II a and Pthese damage responses and the Trapped in Agarose DNA Immunostaining assay was utilised to quantify the numbers of topo II_DNA complexes formed in response to these agents. This study aimed to examine the levels of DNA damage following exposure to these damaging agents and to investigate differences in the complement of proteins associated with DNA damage-induced foci. By using both the y-H2AX and TARDIS assays and protein colocalisation techniques, the studies detailed here presents novel findings on the differing damage responses induced following these three agents.