Tools such as protein immunoblotting have proven benefits for investigating T lymphocyte signaling but have several drawbacks such as the number of cells required and the difficulty of distinguishing subset-specific differences without expensive and invasive cell sorting. Recent advances in immunology and the identification of T lymphocyte sub-populations making up only a very small fraction of the total population highlight the importance of studying signaling in those small subsets in a feasible, cost-effective, high-throughput manner. To this end, we have developed a simplified protocol to study both intracellular phosphorylation patterns of important signal transduction molecules concomitantly with T cell surface marker expression. A multi-parametric analysis may allow the quantification of the phosphorylation of up to five signaling molecules in CD4 and CD8 T lymphocytes and their naïve, central memory, effector memory, and TEMRA subsets. This enables precise identification of subset-specific signaling and alterations of signaling pathways in physiological and pathological situations. The importance of such detailed analysis is discussed.