Detection of nonselective adenoviruses in tissue- or tumor-selective oncolytic adenovirus preparations presents a technical challenge because of the conditionally replication-competent nature of oncolytic adenoviruses. Although quantitative PCR has been used extensively for detecting specific genes that are likely present in nonselective recombinants, the actual biological activity of nonselective genetic recombinants has not been demonstrated. Therefore, a bioassay that amplifies nonselective adenoviruses through multiple passages in nonpermissive cells was developed to detect biologically active nonselective recombinants using CG7870, a prostate-specific oncolytic adenovirus. The assay was sensitive, and its results were consistent with a quantitative PCR assay for four lots of CG7870. CG0070, a pan-tumor oncolytic adenovirus with no detectable wild-type-like recombinants by PCR, was subjected to a variation of this bioamplification assay using two different nonpermissive cell lines to both verify PCR results and assess its genetic stability under selection pressure. No evidence of the presence of biologically active nonselective recombinants was seen in the original material or after serial passaging in nonpermissive cells. Thus, this bioamplification assay is able to detect nonselective recombinants, and its results are consistent with quantitative PCR assays. A modified version of this assay is also useful for assessing the genetic stability of oncolytic adenoviruses that have no PCR-detectable recombinants.