Abstract: OBJECTIVE To establish the homogeneous time-resolved fluorescence(HTRF) detection method of NF-κB on cells inflammatory model, and provide the experiment evidence for high throughput screening of respiratory inflammatory drugs. METHODS Confirmed the extent of phosphorylation of NF-κB through testing the ratio of Total-NF-κB and Phospho-NF-κB by HTRF. Observed the extent of phosphorylation of NF-κB in the different conditions of cell density(10, 25, 50, 100, 150 k per well) of bronchial smooth muscle cells(BSMC) and bronchial epithelial cells(HBE), stimulus factors(TNF-α, LPS), and action time(10, 30, 60 min), built the cell inflammatory model and the detection method of NF-κB by HTRF. RESULTS HBE cells were used, the cell density was 10 k per well, the stimulating factor TNF-α(10 ng·mL^-1) stimulated the cells, and the inflammatory cell model was established by stimulation for 10 min, which could increase the phosphorylation level of NF-κB in the cells to 52.6%. Compared with the normal control group (14.2%), there was a significant increase. CONCLUSION A rapid, specific and simple HTRF method for determining the level of NF-κB phosphorylation in the inflammatory model of cells was established, which provide a basis for the screening of airway inflammatory drugs.