During more than 25 years of application in immunological sciences, ELISPOT has been established as a routine, robust, versatile, and reliable assay. From basic research to clinical immune monitoring, ELISPOT is being used to address the quantification and (to a lesser extent) functional characterization of immune cells secreting different molecules in the context of health and disease, immune intervention, and therapy in humans and other species [Kalyuzhny (Ed.) (2005) Handbook of Elispot: methods and protocols, Vol. 302, Humana Press Inc., Totowa, NJ]. Over the last decade, ELISPOT assays have been increasingly implemented as an immune-monitoring tool in clinical trials [Schmittel et al. J Immunother 23:289-295, 2000; Whiteside Immunol Invest 29:149-162, 2000; Nagata et al. Ann N Y Acad Sci 1037:10-15, 2004; Cox et al. (2005) Cellular immune assays for evaluation of vaccine efficacy in developing countries., In Manual of Clinical Immunology Laboratory (Rose, N. R., Hamilton, R. G., and Detrick, B., Eds.), p 301, ASM Press, Washington, DC; Cox et al. Methods 38:274-282, 2006]. While the principles of the original protocol have changed little since its first introduction [Czerkinsky J Immunol Methods 110:29-36, 1988], individual laboratories have adapted assay procedures based on experimental needs, availability of reagents and equipment, obtained recommendations, and gained experience, leading to a wide disparity of applied ELISPOT protocols with inevitable consequences. This chapter addresses the resulting challenges for ELISPOT use in clinical trial settings, and discusses the influence of harmonization strategies as a tool for overcoming these challenges. Furthermore, harmonization is discussed in the context of assay standardization and validation strategies.