To develop and validate a novel reporter gene assay (RGA) to detect pyrogen, HL60 cells were transfected with an NF-κB-RE plasmid containing the luciferase gene to generate stably transfected cells. Through stimulation with pyrogens, a signal was obtained that was dose-dependent with the concentration of pyrogen. Using the cells, we selected and optimized the parameters and found that the optimal conditions may be with 5 × 105/ml cells that were seeded and incubated with pyrogen for 3-6 h in IMDM medium with 2% FBS. Based on the optimized parameters, a novel RGA was developed. Then, the RGA was validated and the results showed that the linearity was greater than 0.95 between the signals and the concentrations of pyrogen, the recoveries of pyrogen were all between 50% and 200%, and the precision was less than 35%. There was no difference in the sensitivity, specificity or reproducibility between RGA and BET, and the results from RGA and MAT and RPT were consistent. Furthermore, the RGA can be applied to the pyrogen detection of monoclonal antibodies. Due to its advantages including a fast detection speed, high sensitivity, convenient mode of operation and wide-pyrogen spectrum detection, RGA is promising as a supplementary method to detect pyrogen.