The ability to detect and quantify viral vector sequences from a variety of clinical sample types is crucial to performing biosafety risk assessments. Viral vector-mediated gene transfer studies are often performed in animals, and these animals must be placed under appropriate biocontainment conditions to protect the workers and environment. Data on the shedding of viral vectors from animals are limited, and the sample types are challenging for polymerase chain reaction (PCR). For this reason, we developed a quantitative PCR assay that could be used for such purpose. We designed a sequence-specific, probe-binding method for detecting adenoviral and lentiviral vector sequences. A duplex strategy was used that included a quality control sequence to be amplified in the same reaction as the viral vector. This sequence provided an internal control for normalization of noncellular sample types, such as animal excretions that inherently lack a natural control sequence. The new assay was used to establish the efficiency of reverse transcription and to detect viral genomes in stocks of whole virus particles. We identified sets of primers and probes for both adenoviral and lentiviral sequences that work well together with no interference. The average conversion rate of RNA into complementary DNA was 18.5%. The new quantitative PCR assay was efficient and specific, and it measured successfully the number of viral genomes in stocks of whole virus particles. This assay could be used to detect adenoviral and lentiviral vector sequences for biosafety and other research purposes.