An expressed luciferase viability assay (ELVA) has been developed for cell viability and cell number based on detecting the expression of luciferase transfected into the cells. Stable transfectants were produced that expressed luciferase constitutively. Like many endogenous enzymes, luciferase is rapidly degraded following cell death, so that the enzyme can be used as a measure of cell viability. A modified luciferase assay was used in which the reagents were added directly to the cells in a microplate. The main advantages compared to other cell viability assays are the wide dynamic range, high sensitivity, low background, and the absence of any requirement to wash or harvest the cells. Stable transfectants of three factor-dependent cell lines (B13, Ba/F3 and CTLL) were produced and used in cytokine assays. Three strategies of selection after electroporation were tested: (1) using a plasmid containing both the genes encoding firefly luciferase and a selectable marker (neo), (2) cotransfection of a plasmid containing luciferase and a plasmid containing a selectable marker (puromycin resistance), and (3) cotransfection of a plasmid containing luciferase and a plasmid containing the human IL-5Ralpha-chain, and selecting in IL-5. This latter strategy produces an IL-5 responsive cell line expressing luciferase in a single step without the need for antibiotic selection.