The use of replication-competent oncolytic viruses has largely advanced cancer gene therapy. Oncolytic virus not only possesses unique mechanisms of action that are distinct from other treatment modalities, its self-perpetuating nature provides an ideal platform for therapeutic transgene insertion. Tumor selectivity can be achieved by deleting viral genes that are critical for growth in normal cells but dispensable in tumor cells, transcriptional control under tumor-specific promoters, fiber modification targeting tumor-specific cellular receptors, or the use of inherent tumor-specific viruses. Transgene products can be amplified along with viral replication, thus maximizing therapeutic effect. Using adenovirus as a template, this chapter describes common assays used for the study of oncolytic viruses, with special emphasis on in vitro and in vivo viral replication determination.