The transduction efficiency of viral vectors can easily be monitored during pre-clinical trials by inclusion of marker genes. However, the use of such marker genes has to be avoided in the final clinical gene therapy situation since their products often represent powerful immunogens and lead to the elimination of transduced cells. Thus it is not desirable to use such genes as markers in clinical settings, especially if the vector is applied in vivo. In these cases PCR based methods like the real-time PCR might provide a powerful tool to estimate biodistribution. In order to investigate the accuracy and precision of this method, we have developed and tested a real-time PCR assay for the quantification of the enhanced green fluorescent protein (EGFP) gene and compared the results with transduction efficiencies estimated by FACS analysis. Although the real-time PCR technique itself is characterized by low inter- and intra-assay variations over a wide dynamic range of quantification, significant differences in the transduction efficiency compared to FACS data have been observed. To compensate for this, an optimized multiplex real-time PCR assay, which allows the simultaneous calculation of cell number and EGFP copy number in the same tube was implemented. In view of future needs for methods allowing the accurate analysis of biodistribution in gene therapy trials our data highlights the necessity to rigorously check the accuracy and precision of the assay system used.