The FluoroSpot assay is an inventive development of the ELISpot assay and uses fluorescent detection instead of colorimetric. This allows for simultaneous detection of several analytes, which is a major advantage. The human IFN-γ/Granzyme B FluoroSpot kit enables convenient enumeration of cells secreting either one or both analytes. The kit contains low fluorescent plates, matched quantities of monoclonal capture and detection antibodies, secondary reagents conjugated to fluorophores, a fluorescence enhancer and an anti-CD28 monoclonal antibody for co-stimulation. A polyclonal activator for human T cells (mAb CD3-2) is supplied as a positive control for cytokine secretion.