MHC class II tetramers have emerged as an important tool for characterization of the specificity and phenotype of CD4 T cell immune responses, useful in a large variety of disease and vaccine studies. Issues of specific T cell frequency, biodistribution, and avidity, coupled with the large genetic diversity of potential class II restriction elements, require targeted experimental design. Translational opportunities for immune disease monitoring are driving the rapid development of HLA class II tetramer use in clinical applications, together with innovations in tetramer production and epitope discovery.