This fourth edition volume expands on the previous editions with discussions on the latest prools that can be applied in different laboratory landscapes ranging from purely research to clinical diagnostic environments. This volume particularly focuses on ELISpot validation and evaluation of antibodies used in this assay. The chapters in this volume cover topics such as peptides in T-Cell assays and ELISpot applications for monitoring infectious diseases like tuberculosis and SARS-CoV2; FluoroSpot; the use of AI for studying ELISpot; and comparing ELISpot with flow cytometry, feline coronavirus (FCoV2), and memory b-cells. Written in the highly successful series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory prools, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, is a valuable resource to aid in the advancement of science and technology, and inspire scientists and researchers to determine and pursue new discoveries in this developing field. Chapters 5, 7, 11, 13, and 15 are available open access under Creative Commons Attribution 4.0 International License via link.springer.com.