The mammalian innate immune system provides a first line of defense against microbial pathogens and also serves to activate an antigen specific acquired immune program. Key components of innate immunity are the interferons (IFNs), a family of related cytokines with potent antimicrobial and immuno-modulatory activities. The IFNs exert their effects through the induction of numerous genes, one of which is the double-stranded RNA-dependent protein kinase (PKR), a pivotal antiviral protein found in most human cells. Following activation by double stranded (ds) RNAs produced during viral replication, PKR phosphorylates the α-subunit of eukaryotic translation initiation factor (eIF) 2, causing a severe inhibititon of cellular and viral protein synthesis. Phosphorylation of eIF2α and consequent inhibition of protein synthesis is a major cell growth checkpoint utilized by at least three other kinases, in addition to PKR, following exposure to such cellular stresses as amino acid deprivation and the presence of misfolded proteins in the endoplasmic reticulum. Indeed, it has been demonstrated that disruption of the eIF2α checkpoint can lead to the transformation of immortalized rodent and human cells, plausibly by increasing the protein synthesis rates of proto-oncogenes. Further, it has been shown that disregulation of the eIF2α checkpoint and consequent permissiveness to virus infection may be a common occurrence in tumorigenic mammalian cell lines. These findings have been exploited to develop potent oncolytic RNA viruses that can selectively replicate in and destroy a variety of neoplasias in vitro and in vivo. In this chapter, we describe some of the techniques commonly used in our laboratory to examine PKR activity and eIF2 regulation. Protocols for the generation and use of recombinant vesicular stomatitis virus variants are also described.