Ideally, gene transfer vectors used in clinical protocols should only express the gene of interest. So far most vectors have contained marker genes to aid their titration. We have used quantitative real-time PCR to titrate equine infectious anemia virus (EIAV) vectors for gene therapy applications. Viral RNA was isolated from vector preparations and analyzed in a one-step RT-PCR reaction in which reverse transcription and amplification were combined in one tube. The PCR assay of vector stocks was quantitative and linear over four orders of magnitude. In tandem, the integration efficiency of these vectors has also been determined by real-time PCR, measuring the number of vector genomes in the target cells. We have found that these methods permit reliable and sensitive titration of lentiviral vectors independent from the expression of a transgene. They also allow us to determine the integration efficiency of different vector genomes. This technology has proved very useful, especially in the absence of marker genes and where vectors express multiple genes.