Feline leukemia virus (FeLV) is a gamma retrovirus that induces fatal diseases in domestic cats. Efficacious FeLV vaccines prevent persistent viremia and development of FeLV-related disease after virus exposure, but not minimal viral replication and a provirus-positive state as recently demonstrated using sensitive real-time PCR assays. Proviral integration is an important parameter of latent infection and persistence of retroviruses in infected cells. So far, FeLV-specific real-time PCR assays could not distinguish between the integrated and episomal forms of the provirus. Thus, it was the aim of the present study to develop a rapid assay for the detection of FeLV proviral integration. The test combines conventional and quantitative real-time PCR that use virus-specific primers and primers specific for cat genomic small interspersed nuclear elements. It was applied to analyze the time course of proviral integration into the genome of a feline fibroblast cell line and detect provirus integration in peripheral white blood cells from vaccinated and unvaccinated, FeLV-exposed cats. The newly developed rapid test will essentially contribute to a better understanding of the mechanisms involved in the pathogenesis of FeLV infection and be especially useful in the development of antiretroviral vaccines and therapies aimed at the inhibition of proviral integration.