Abstract: Methods and compositions are provided for analyzing in viable cells gene expression regulatory elements and systems. The method can be used to evaluate a transcriptional initiation regulatory system or the presence of a trans acting component. The method finds use in evaluating various transcriptional initiation functional sequences for use in expression of products, for the detection of trans acting agents associated with viruses and for quantitating the presence of infectious viruses. The method comprises introducing an expression cassette encoding an enzyme which has a fluorescent substrate, substantially irreversibly introducing the substrate into the cell host and detecting the formation of fluorescence during a predetermined time interval.