The CellTiter-Fluor Cell Viability Assay is a non-lytic, single-reagent-addition fluorescence assay that measures the relative number of viable cells in a population. The assay is based on measurement of a conserved and constitutive protease activity within live cells and therefore serves as a biomarker of cell viability. The live-cell protease activity is restricted to intact viable cells and is measured using a fluorogenic, cell-permeant, peptide substrate (Gly-Phe-AFC). The substrate enters intact cells, where it is cleaved by the live-cell protease activity to generate a fluorescent signal proportional to the number of living cells. The live-cell protease becomes inactive upon loss of membrane integrity and leakage into the surrounding culture medium. The CellTiter-Fluor Assay also can be used in a single-well, sequential, multiplex format with other downstream assay chemistries to normalize data by cell number. Data from the assay can serve as an internal control and allow identification of errors resulting from cell clumping or compound cytotoxicity. The assay is compatible with many Promega luminescence assays or spectrally distinct fluorescence assay methods, such as measuring caspase activation, reporter gene expression or orthogonal measures of viability.