During the last 20 yr, the enzyme-linked immunospot (ELISPOT) assay has emerged as one of the most important and widely used assays to monitor immune responses in humans and a variety of other species. With the ELISPOT assay, immune cell frequencies can be measured at the single cell level without elaborate expansion or manipulation of cell populations. Its usefulness has led to its application in vaccine design and development and, most importantly, in monitoring vaccination efforts. The impact of results measured with this assay can be profound. In addition to ease of performance, repeatability and reliability are major features expected of an ELISPOT assay. The focus today is on standardization of the technique, validation strategies to comply with these required features, and accommodation of the growing demand of Good Laboratory Practice (GLP) compliance. This chapter will give the experienced scientists as well as newcomers to the field an overview over the major standardization issues for each step of the protocol. Guidelines are given on how to validate the ELISPOT performance.