T cell activation plays a central role in inflammation, autoimmune diseases and cancer. Cancer immunotherapies, such as immune checkpoint inhibitor, bi-specific antibody, chimeric antigen receptor T (CAR T) cell, and adoptive tumor-infiltrating lymphocyte (TIL) therapies require the characterization and monitoring of T cell activation. Here we describe a novel, multiplex immune assay platform based on high-throughput flow cytometry technology and advanced computational algorithms for data analysis. The assay simultaneously measures T cell dynamics including phenotype, time-dependent expression of activation markers, secreted effector cytokines, and proliferation. The assay screened a kinase chemogenomic library and identified 25 kinase inhibitors with distinct inhibition profiles on early (CD69) and late (CD25) activation markers and the cytokines IFNγ and TNFα. We identified 5 kinase inhibitors with dissimilar effects on CD69 and CD25 expression, and a cluster of total 4 MEK1//2 inhibitors with similar activation profiles. The screening revealed 3 kinase inhibitors for PKC, IKK2, and MEK1/2 respectively, all with a phenotypic signature similar to ruxolitinib, a Jak1/2 inhibitor used to treat myelofibrosis disease. These results suggest this multiplexed assay platform, combined with a chemogenomic library screening, may be used as primary screen for phenotypic or target-based drug discovery, target identification, and potential drug repositioning.