Vector biodistribution and clearance studies are essential in the development of gene transfer medicine. To provide reliable and accurate data, protocols for vector analysis must be optimized and validated. We addressed several parameters affecting the detection of gene therapy vectors in blood. Using an in vitro system based on plasmid DNA incorporating, as a transgene, complementary DNA for human erythropoietin gene, we developed and validated a suite of real-time PCR assays for the transgene splicing sites. The most sensitive assays detected the transgene present at 0.011% of the copy number of the endogenous erythropoietin gene in human genomic DNA at 100% specificity. Plasmid linearization incorporated with PCR resulted in an increase in assay sensitivity up to 4.5-fold without compromising analysis workflow. This allowed detection of five copies of transgene in a background of 0.4 μg of genomic DNA (or 0.0035% detectable transgene copies relevant to copies of the endogenous gene). Finally, desktop assessment of 18 DNA extraction protocols was undertaken and 5 kits were evaluated experimentally for extraction of nonviral vectors from blood. Three kits reliably detected 80 copies of the transgene in a milliliter of blood. Adoption of the described protocols will enable more reliable vector analysis in gene therapy and will assist in accurate interlaboratory comparison. The methodology will also facilitate detection of gene doping in sport, a potential new form of misuse of gene transfer technology.