Enzyme-linked immunospot (ELISPOT) is an assay used to detect secretion of cytokines from immune cells. The resolution and sensitivity of ELISPOT allow for the detection of rare T cell specificities and small quantities of molecules produced by individual cells. In this chapter, we describe an epitope screening method that uses CD4+ T cell ELISPOT assays to identify specific novel mycobacterial antigens as potential vaccine candidates. In order to screen a large number of candidate epitopes simultaneously, pools of predicted MHC class II peptides were used to identify mycobacterial specific CD4+ T cells. Using this method, we identified novel mycobacterial antigens as vaccine candidates.