The present study is about diagnosis of IRAK-4-deficiency by flow cytometric measurement of IκB-α degradationA flow cytometric assay that is able to detect aberrant NF-κB signaling in IRAK-4-deficient patients was developed.The test is based on evaluation of IκB-α degradation in monocytes with or without stimulation with IL-1β, TNF-α, PAM3CSK4 (TLR1/2 agonist) and R-848 (TLR7/8 agonist).IκB-α degradation was induced by TNF-α, Pam3CSK4, R-848 and to a lesser extent by IL-1β.Based on these results, following cut-off values for the ratios were determined; 0.98 for IL-1β, 0.62 for TNF-α, 0.61 for PAM3CSK4 and 0.65 for R-848.IRAK-4-deficient monocytes showed significantly less IκB-α degradation compared to control monocytes after stimulation with IL-1β, PAM3CSK4 and R-848.The ratio of the MFIs in stimulated monocytes to unstimulated monocytes was (mostly) above the cut-off values after stimulation with these ligands.No significant difference was seen between stimulated NEMO-deficient monocytes and stimulated control monocytes.However, 1 NEMO-deficient patient showed a ratio just above the cut-off values after stimulation with TNF-α, Pam3CSK4 and R-848.In conclusion, assessment of IκB-α degradation by flow cytometry in monocytes provides a rapid and useful tool to reveal aberrant NF-κB signaling in IRAK-4-deficient patients.Although we did not have access to MyD88-deficient patients, we expect that the results will be similar (as MyD88 is just upstream of IRAK-4 in the TLR pathway).Moreover, others have shown impaired IκB-α degradation in IκB-α mutated fibroblasts and in CARD11-deficient B and T lymphocytes upon stimulation with PMA/ionomycin.Further investigation is needed to expand the use of this test to other ligands and other inborn errors of immunity caused by defects in the canonical NF-κB pathway.