T follicular helper (T(FH)) cells stimulate, whereas T follicular regulatory (T(FR)) cells inhibit, B cell responses. Despite the potent immunoregulatory roles for TFR cells in controlling the magnitude of antibody production, the precise mechanisms by which T(FR) cells exert their suppressive effects are not yet clear. The lack of specific assays to assess T(FR) cell function separately from differentiation has hindered progress in elucidating T(FR) cell function. This is due, in part, to difficulty in separating T(FR) cells from phenotypically similar, but functionally different, T(FH) cells. Here we describe an in vitro approach for sensitively and quantitatively assessing the capacity of T(FR) cells to suppress T(FH)-mediated B cell antibody production utilizing both ELISA and flow cytometry to measure B cell responses. Beyond assessing T(FR) function, this assay system can also be used to sensitively measure T(FH) stimulatory capacity as well as B cell function.