The red-shifted S65T mutant green fluorescent protein (GFP) was used to compare the adenovirus (Ad) production and post-infection survival of 293SF and 293S cells in serum-free and serum-containing flask cultures, respectively. The GFP-expressing vector permitted the quantification of both the level of GFP expressed by infected cells and the infectious viral content of the cultures by flow cytometry in a simple, fast, sensitive, and reliable way. The GFP has the main advantage of fluorescing without any substrate addition. Infected cultures showed the coexistence of two populations of fluorescent cells, high-fluorescence cells (HFCs) and low-fluorescence cells (LFCs), in proportions that varied between 20 and 75 hpi. The gradual increase in the number of LFCs at the expense of HFCs correlated well with the increase in the number of dead cells. This relationship could be used for the continuous measure of a culture’s viability with the appropriate on-line instrumentation. The post-infection death rate of infected 293SF cells was higher than that of infected 293S cells, but the level of GFP fluorescence in viable, highly fluorescent cells was similar in the two infected cell lines. The number of infectious viral particles (IVPs) was quantified in less than 24 h by an infection assay of 293S cells in wells with viral particles extracted from the culture samples, and the results were more reproducible (+/-10% variation) than those generally reported for conventional plaque assay titrations or end-point dilutions. The viable cell-specific IVP concentrations were for most experiments similar, indicating again that the difference between the two cell lines was their unequal post-infection viabilities, not the virus production by the infected living cells.