The HCS LIVE⁄DEAD® Green Kit measures cytotoxicity by high content analysis with the Image-iT® DEAD Green™ viability stain – a non-fluorescent and cell impermeant compound that exhibits a strong fluorescent enhancement upon binding to DNA. Drugs and test compounds leading to serious cell injuries, including plasma membrane permeability, allow entry of the Image-iT® DEAD Green™ viability stain. This two-color fluorescence assay also employs the use of a cell permeant nuclear segmentation tool that stains both live and dead cells, reflecting the total cell number within the sample. For total cell staining, the kit includes both a blue-fluorescent Hoechst 33342 and a near infrared-fluorescent, HCS NuclearMask™ Deep Red reagent. Either of these segmentation tools may be used with Image-iT® DEAD Green™ viability stain for excellent discrimination of live and dead cells that survives fixation and permeabilization for combination with additional parameters of cytotoxicity or other biomarkers. The kit contains sufficient reagents for performing 2, 96-well plates.