The MultiTox-Glo Multiplex Cytotoxicity Assay is a sequential-reagent-addition fluorescent and luminescent assay that measures the relative number of live and dead cells in cell populations. The MultiTox-Glo Assay sequentially measures two protease activities; one is a marker of viability, and the other is a marker of cytotoxicity. The live-cell protease activity is restricted to intact viable cells and is measured using a fluorogenic, cell-permeant, peptide substrate (GF-AFC). This substrate enters intact cells, where it is cleaved by the live cell protease activity to release AFC and generate a fluorescent signal that is proportional to the number of viable cells. The live-cell protease becomes inactive upon loss of membrane integrity and leakage into the surrounding culture medium. A second, luminogenic cell-impermeant peptide substrate (AAF-aminoluciferin) is used to measure dead-cell protease activity, which is released from cells that have lost membrane integrity. The liberated aminoluciferin product is measured as glow type luminescence generated by Ultra-Glo Recombinant Luciferase provided in the assay reagent. The MultiTox-Glo Assay gives ratiometric, inversely correlated measures of cell viability and cytotoxicity, which correlate with established methods for measuring viability and cytotoxicity. The ratio of viable cells to dead cells is independent of cell number and, therefore, can be used to normalize data. Having complementary cell viability and cytotoxicity measures reduces errors associated with pipetting and cell clumping, as well as serving as an internal control to allow identification of errors resulting from chemical interference from test compounds or media components.