A biological assay for detecting a non-selective replication competent virus (RCV) in a preparation of selectively-replicating virus. The method comprises -passaging the virus preparation at least once on cells that differentially amplify the selectively-replicating virus versus a replication competent virus and further analyzing the virus preparation, thereby detecting the presence of a replication competent virus. The further analysis of the virus preparation includes one or more of the following: detecting increased cytopathic effect; detecting increased virus production; detecting increased potency to kill normal cells; detecting an altered restriction digest pattern; detecting an altered viral genome sequence; detecting anticipated recombinants with PCR amplification; and detecting acute hepatotoxicity in an animal administered the virus preparation. In one embodiment, the assay detects replication competent adenoviruses in a preparation of an oncolytic adenovirus.
