Phosphoflow cytometry is increasingly being used as a tool for the discovery of biomarkers used in the treatment and monitoring of disease and therapy. The ability to measure numerous phospho-protein targets simultaneously at a single cell level accurately and rapidly provides significant advantages over other methods. We here discuss important considerations required to successfully implement these methods. Three different blood collection tubes (lithium-heparin tubes, CPT with sodium citrate and CPT with sodium heparin) were evaluated, with PBMC isolated through lithium-heparin tubes/lymphoprep displaying reduced basal and increased stimulation induced phosphorylation compared to the other two methods. Further, we provide a protocol outlining an 8 color assay developed for the study of intracellular signaling in peripheral blood mononuclear cells. The assay allows for the quantitative measurement of the phospho-proteins ERK1/2, NF-?B p65, Stat1 (Y701), Stat1 (S727), Stat3 (Y705), Stat3 (S727), Stat4 (Y693), p38 and Stat5 (Y694), as well as the identification of T cells, B cells, natural killer cells and monocytes. The assay additionally incorporates fluorescent cell barcoding, reducing assay costs and increasing throughput while increasing data robustness. Inter-assay precision was assessed over a month long period for all experimental variables (phospho-protein measured, cell type and stimulant). Coefficient of variations (CVs) calculated from process triplicates of normalized median fluorescence intensity (MFI) of the phospho-proteins displayed median CVs under 10% when grouped according to cell type, stimulation agent and phospho-protein measured, while the CV for each triplicate did not exceed 20%.