After the life-threatening cytokine release syndrome (CRS) occurred in the clinical study of the anti-CD28 monoclonal antibody (mAb) TGN1412, in vitro cytokine release assays using human blood cells have been proposed for non-clinical evaluation of the potential risk of CRS. Two basic assay formats are frequently used: human peripheral blood mononuclear cells (PBMC) with immobilized mAbs, and whole blood with aqueous mAbs. However, the suitability of the whole blood cytokine assay (WBCA) has been questioned, because an unrealistically large sample size would be required to detect the potential risk of CRS induced by TGN1412, which has low sensitivity. We performed a WBCA using peripheral blood obtained from 68 healthy volunteers to compare two high risk mAbs, the TGN1412 analogue anti-CD28 superagonistic mAb (CD28SA) and the FcγR-mediated alemutuzumab, with a low risk mAb, panitumumab. Based on the cytokine measurements in this study, the sample size required to detect a statistically significant increase in cytokines with 90% power and 5% significance was determined to be n = 9 for CD28SA and n = 5 for alemtuzumab. The most sensitive marker was IL-8. The results suggest that WBCA is a practical test design that can warn of the potential risk of FcγR-mediated alemtuzumab and T-cell activating CD28SA but, because there was apparently a lower response to CD28SA, it cannot be used as a risk-ranking tool. WBCA is suggested to be a helpful tool for identifying potential FcγR-mediated hazards, but further mechanistic understanding of the response to CD28SA is necessary before applying it to T cell-stimulating mAbs.