The enzyme-linked immunospot (ELISPOT) assay is a sensitive and relatively simple assay for detecting secreted cellular products such as cytokines and has become an invaluable immunological tool. The ELISPOT has been used extensively in human and murine research but has only recently been used to assess the feline immune system. For researchers studying feline disease or using the cat as a model of human disease, the quantification of cytokine-producing cells by ELISPOT is an invaluable technique for investigations of disease immunopathogenesis and vaccine efficacy. For example, use of the interferon (IFN)-gamma ELISPOT to measure the frequency of antigen-specific T-cells during feline immunodeficiency virus (FIV) infection or after immunization with candidate FIV vaccines is of particular interest. This application of the ELISPOT may serve to expand the utility of FIV as a model for human immunodeficiency virus. Broader applications of the ELISPOT should further our understanding of feline diseases and be useful in the rational development of more efficacious vaccines and therapeutic modalities for the enhancement of feline health. This chapter discusses important parameters of ELISPOT design that will enable researchers to develop and analyze the feline-specific assays within their own laboratory.