Since the first report of an engineered oncolytic virus, there has been a continuing and steady increase of interest in the field, and while bench research remains vital for the translation of research in this field, its success depends on breakthroughs in clinical studies. Oncolytic Viruses: Methods and Protocols describes the construction and purification of capsid-modified adenoviruses as well as oncolytic adenoviruses, presents protocols for many individual virus species including engineering and preparation of oncolytic HSV, propagation, purification, and in vivo testing of oncolytic VSV, details properties of oncolytic reovirus and NDV, and describes the generation and testing of next generation of oncolytic vaccinia virus. As the host immune system plays a critical role in determining efficacy of oncolytic viruses, two chapters are devoted to the study of immune response. Recent advances in stem cell research have led the field in two distinct directions: the use of stem cells as carrier vehicles for oncolytic viruses and the targeting of cancer stem cells. As such, the volume describes the use of explant tissue samples from patients to potentially provide useful information predicting responses prior to clinical translation. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Oncolytic Viruses: Methods and Protocols seeks to serve both professionals and novices with its well-honed methodologies in an effort to further our knowledge of this essential and vital field.