The enzyme-linked immunospot (Elispot) assay is a widely used technique to monitor cells on the single cell level for the release of analytes like cytokines, chemokines or immunoglobulins (antibodies), in response to particular stimuli. The most important feature of Elispot is its outstanding sensitivity, allowing the detection of specific cells in very low frequencies. The advantages of Elispot have resulted in it being widely adapted for use in research and translational applications in numerous fields including cancer, infectious diseases, autoimmunity, and transplantation.