The enzyme-linked immuno-spot (ELISPOT) technique is a sensitive method used for measurement of elusive immune markers in limited-volume samples. By virtue of the exquisite sensitivity of the ELISPOT assay, frequency analysis of rare cell populations (e.g., antigen-specific responses), which was not possible before, is now relatively easy. However, development of a method sensitive enough to pinpoint elusive immune markers at the single-cell level is a challenge since there are a number of demands that have to be fulfilled and traps to avoid, achieving a valuable outcome.To optimize the environment for in vitro culture and analysis of immune spots by ELISPOT, a number of criteria have to be fulfilled: processing of sample and perhaps also cryopreservation of cells before analysis and, for the ELISPOT assay, optimal cell culture, positive and negative controls, antigen concentration, and, finally, development and readout of spots.If these criteria are fulfilled for your ELISPOT assay, you will likely have the opportunity to pinpoint elusive immune markers at the single-cell level. This chapter describes the ELISPOT assay for detection of cytokines (e.g., IFN-γ and IL-4), with focus on the main criteria that affect the assay. However, this method could be easily adapted to measure other immune markers in small volumes of biological samples.