This protocol describes the preparation and use of ELISpot 96 well plates for measuring human IFNγ and Granzyme B production by immune cells (notably CD4 and CD8 T cells). Cells harvested from peripheral blood or other organs can be used, stimulated with antigen of interest (usually between 18 to 24h), and per cell production of IFNγ and Granzyme B measured as a ‘spot’ on a plate well membrane (http://www.immunospot.eu/advantage-of-immunospot-kits.html).