Enzyme-linked immunospot, or ELISPOT, assay allows the detection of low frequencies of cells secreting various molecules. ELISPOT can be used in many areas of research and, because of its high sensitivity, has the potential to become a valuable diagnostic tool. Based on the same “sandwich” immunochemical principles as enzyme-linked immunosorbent assay, ELISPOT is easy to perform and quantify the results. At the same time ELISPOT remains a state-of-the-art technique that requires accuracy, thorough selection of antibodies and detection reagents, and an understanding of the principles of data analysis. This review covers various technical aspects of the ELISPOT assay, including immunochemical principles of the assay, selection of reagents and plates, and troubleshooting recommendations.