Abstract Peripheral Blood Mononuclear Cells (PBMC) are composed of all blood cells having a round nucleus. This includes lymphocytes, monocytes, and macrophages. The lymphocyte population is made up of T cells, B cells, and Natural Killer cells. Th17 cells are recently discovered T helper cell subtype that plays an important role in the establishment and maximization of the capabilities of the immune system. Th17 cells are derived from CD4+ lymphocytes which are prevalent in human PBMC samples. In this study, we used several multiplex cell signaling immunoassay panels and a human Th17 cytokine multiplex immunoassay panel to compose a biochemical profile of PBMCs before, during, and after a Th17 differentiation process. We also examined the effect of serum starvation and refeeding on undifferentiated and differentiated PBMC. Our differentiation process consisted of a 7-day treatment of four cytokines, IL-1β, IL-6, IL-23, and TGFβ. Cell signaling panel results demonstrated that several analytes exhibited increased constitutive expression and phosphorylation after differentiation and stimulation with serum. Additionally, the human Th17 cytokine panel detected a significantly increased secretion of many cytokines including Th17 cell specific cytokines from human PBMCs (e.g. IL-17F). In conclusion, the Th17 and cell signaling panels will be useful tools for Th17 cell-related cytokine and signaling profiling in PBMC and various biological samples.