Recently, new methods have been introduced describing assessment of antigen-specific CD4+ T-cell immunity according to the induction of CD154 (CD40L) on CD4+ T cells during short-term activation. In our study, we have evaluated the influence of different stimulation conditions on the flow cytometric analysis of CD154 expression after antigenic in vitro activation. We used different cell preparation methods, antigen sources, and time periods of in vitro stimulation and analyzed their impact on intra and extracellular detection of antigen-induced CD154 expression on CD4+ T cells. We could demonstrate that analysis of CD4+ T-cell immunity according to CD154 expression displayed low intra-assay variability and was robust with respect to its induction in the course of a variety of stimulation conditions. For a basic quantitative evaluation of antigen-specific CD4+ T cells, surface CD154 analysis could be employed, enabling the fast analysis of live antigen-specific CD4+ T cells. Intracellular analysis of CD154 in combination with cytokines such as IL-2 and IFNgamma allowed quantitative and qualitative assessment of antigen-specific CD4+ T cells. The cytometric analysis of antigen-specific CD4+ T-cell immunity according to CD154 expression is characterized by robustness, high sensitivity, and low intra-assay variability.