T follicular helper (Tfh) cells are a subset of CD4(+) T cells that accumulate in the B cell-rich regions of secondary lymphoid organs and provide activation signals essential for long-lived humoral immunity. Herein, we describe a flow cytometric cell-based approach to identify Tfh cells within the total leukocyte population isolated from the spleen, lymph nodes, and Peyer's patches of mice. This protocol focuses on markers that have established relevance in Tfh cell differentiation and function allowing its use across varied settings of infection and immunity.