T-follicular helper (Tfh) cells have emerged as an independent CD4(+) helper T-cell lineage required for antigen-selected germinal center B-cell survival, class switch recombination, and differentiation into long-lived plasma cells. The quantification and function of Tfh subsets are currently extensively explored in humans with infectious diseases, cancer, or autoimmune disorders. Reliable methods to identify and isolate human Tfh cells in patients and healthy donors are necessary to perform these studies. Here, we propose a classical and robust flow cytometric method to detect and isolate Tfh cells from human secondary lymphoid organs based on the expression of CXCR5, PD-1, and CD25 in the CD4(+) T-cell population. An alternative protocol using anti-ICOS and anti-Bcl-6 antibodies and requiring fixation and permeabilization steps without a decrease of detection of membrane markers is also described.