Transduction efficiency can be easily monitored during pre-clinical trials by inclusion of marker genes. However, the use of such marker genes should be avoided in the final clinical gene therapy application since their products are often immunogenic, making it difficult to monitor transduction, 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. To investigate the accuracy 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 our real-time PCR assay itself was characterized by a high precision over a wide dynamic range of quantification, significant differences in the transduction efficiency compared with FACS data were initially observed. Accurate determination could only be achieved using an optimized multiplex real-time PCR assay, which allows the simultaneous calculation of cell number and EGFP copy number in the same tube. In view of future needs for methods allowing precise and accurate analysis of biodistribution in gene therapy trials, our data highlight the necessity critically to check both parameters in the implemented assay.