TNF is a key cytokine in autoimmune diseases like rheumatoid arthritis, and TNF antagonists are commonly prescribed therapeutics. Although anti-TNF drugs have enabled a very significant progress in this field, disease heterogeneity remains and causes diversity in patient response. These challenges increase the need for anti-TNF characterization tools that may open perspectives toward the development of personalized medicine. In this study, we present a novel whole blood-based flow cytometry functional assay that allows, within a given whole blood sample, the characterization of an anti-TNF molecule mechanisms of action. Whole blood from healthy human donors was employed to mimic the physiological state but also to streamline experimental workflows. Samples were incubated with LPS alone or in combination with various anti-TNF molecules such as adalimumab (ADA), etanercept (ETA), and infliximab. A 10-color flow cytometry panel including CD69, transmembrane TNF, CD16, CD62L, CD66b, CD11b, and CD54 as activation markers was used following a centrifugation-free protocol. CD69 expression decreased on NK, NKT, and T cells upon treatment with ADA, ETA, and IFX as a direct indication of forward signaling neutralization. Percentages of transmembrane TNF