The HCS DNA Damage kit simultaneously and quantitatively measures two important cell-health parameters: DNA damage and cytotoxicity. DNA damage is detected using an antibody against phosphorylated H2AX (Ser139) which is induced in response to double-strand break (DSB) formation. To detect cytotoxicity, the Image-iT® DEAD™ Green is an impermeant dye in healthy cells that becomes permeant when the plasma membrane of cells is compromised. Both indicators are amenable to fixation and permeabilization, thus allowing for multiplexing with other biomarkers of interest. The blue-fluorescent nuclear segmentation tool Hoechst 33342 is also included and stains DNA in both live and dead cells.