The direct assessment of T helper (T_H)-cell responses specific for antigens is essential to evaluate pathogenic and protective immunity. Presently, analysis and isolation of antigen-specific T_H cells is restricted to cells that produce cytokines, or can be performed only with a rare selection of specific peptide major histocompatibility complex class II (MHC II) multimers. Here we report a new method that enables the assessment and isolation of T_H cells specific for a defined antigen according to CD154 expression induced after stimulation in vitro. We show that antigen-induced CD154 expression is highly sensitive and specific for human and mouse antigen-specific T_H cells. Moreover, the isolation of antigen-specific CD154⁺ T_H cells necessitates only surface staining with antibodies, thereby enabling the fast generation of antigen-specific T_H cell lines. Our approach allows assessment of T_H cells with a defined specificity for the combined quantitative and qualitative analysis of T_H-cell immunity as well as for the isolation of specific T_H cells for targeted cellular immunotherapies.