Flow cytometry is combined with highly specific fluorophore-conjugated antibodies that will only bind to the activated forms of molecules. The advances in flow cytometry enable to perform quantitative multiplexed analysis of single cells within heterogeneous populations stained with specific antibodies for phenotyping in conjunction with antibodies to phosphorylated, i.e., activated molecules within signaling pathways. By reactivating signaling pathways in vitro it is possible to collect data on the responsive state of complex cell populations such as immune cells. In this protocol, peripheral blood mononuclear cells (PBMC) are stimulated with cytokines for the indicated time in a 37°C/CO₂ incubator, fixed immediately with paraformaldehyde to freeze signaling, permeabilized with methanol, and then stained simultaneously with an antibody cocktail to signaling molecules within the JAK-STAT pathway and phenotypic markers for T-cells and B-cells. The protocol shows a basic four-color method which can be expanded to potentially study any signaling pathway in a defined cell subset.