Tuberculosis is a global health problem. The Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccine has variable efficacy (0-80%) so there is a drive to develop novel vaccines. The cytokine, interferon gamma (IFNγ), is an essential component of the protective response to M. tuberculosis (M. tb) infection and is also produced in response to BCG vaccination. Induction of an IFNγ response is used as a biomarker of successful vaccination in the assessment of new tuberculosis (TB) vaccines. The IFNγ ELISPOT assay provides an important tool for TB research. It is used for both the diagnosis of infection (T.Spot assay), and for the evaluation of the immunogenicity of new TB vaccine candidates in human clinical trials, in the non-human primate (NHP) model of TB infection studies. The ELISPOT assay captures IFNγ produced by peripheral blood mononuclear cells (PBMCs) following specific stimulation, onto a membrane so individual cells can be enumerated and the frequency of responding cells determined. Hence spot forming units (SFU) per 106 cells provide the traditional measure for ELISPOT assays. The discriminatory power of SFU is limited. In some situations, the number of SFU in BCG vaccinated, and unvaccinated, subjects was found to be similar, although the spots were observed to be larger in vaccinated subjects. Spot size potentially provides a measure of the quantity of cytokine produced by individual cells. The AID ELISPOT plate reader software used to determine frequency of spots also has the capability to determine the size of each spot. Consideration of spot size in combination with spot forming units was investigated in our studies of BCG immunogenicity. This additional readout was found to enhance the discriminatory power of the ELISPOT assay, and provide more information on the immune response to BCG vaccination and infection with M.tb.