The production of high-titer recombinant retrovirus is a major determinant of the efficiency of target cell transduction. Titer assessment for producer clones that contain vectors encoding proteins that can be detected using fluorescence is typically performed by flow cytometry. However, this method is both costly and labor intensive, severely limiting the number of clones that can be screened for each construct. In this report we describe a rapid, high-throughput screening method for viral quantitation of producer clone supernatant on target cells using a 96-well format. Plates were assayed using a multichannel fluorescent reader to determine the percentage of target cells expressing green (EGFP), cyan (ECFP), yellow (EYFP) or red (DsRed) fluorescent reporter genes, or their combinations. The relative fluorescence counts of target cells incubated with viral supernatant from each packaging cell clone correlated with the level of transduction, and hence, viral titer. Correlation of cell fluorescence between the fluorescent plate reader assay and flow cytometric assessment was high (r(2) = 0.96). Independent detection of different fluorescent reporters enabled multiplex assays to be performed. Simultaneous cell density analysis using alamarBlue fluorescence was proportional to cell number per well (r(2) = 1.0). In situ titer assessment of 66 FLYRD packaging cells encoding the EGFP reporter gene identified clones (>10(7) colony forming units per milliliter [CFU/ml]) that provided titers up to sevenfold over the parent population. The application of this rapid, high-throughput screening method overcomes many limitations imposed by the current flow cytometric screening method. This robust assay maximizes the chance of identifying rare high-titer packaging clones and offers a further opportunity to optimize gene transfer protocols.