Mucosa-associated invariant T (MAIT) cells are a relatively recently described subset of innate-like T cells that recognize bacteria- and fungi-derived vitamin B2 metabolites presented by the evolutionarily conserved MHC class-like MR1 molecules. The study of MAIT cells is still in its infancy and methodology needs to be developed to determine their role in human immune defense and disease. In this study, we have established protocols to examine a range of MAIT cell functions as they respond to antigens produced by Escherichia coli. Our optimized experimental protocols allow detailed studies of MAIT cell activation and proliferation in response to E. coli-pulsed antigen presenting cells, as assessed by expression of activation markers, production of cytokines, and dilution of a proliferation tracing dye, respectively, as well as of MAIT cell cytotoxicity against target cells. Given the innate-like characteristics of MAIT cells and the novel type of antigens they recognize, the methodology here developed opens new possibilities to study MAIT cell immunobiology and to determine their role in a range of human disease settings using E. coli as a model antigen source.