ObjectiveTo explore the most suitable reaction condition for human leukocyte elastase (HLE) and its substrates, so as to establish a high-throughput in vitro screening model for HLE inhibitor. MethodsBy measuring the UV absorbance of the product p-NA, we studied the electrolyte concentration, temperature, pH, enzyme/substrate concentration, the influence of reaction time, and detection wavelength on the reaction, so as to determine the optimal condition for the reaction between HLE and its substrates. ResultsThe suitable reaction condition was determined as follows: HLE (0.2 U/ml) 30 μl, substrate (50 mmol/L) 100 μl, Tris buffer(1.0 mol/L NaCl, pH 7.5), temperature 37℃, reaction time 24 h, and detection wavelength 545 nm. ConclusionWe have successfully established a stable in vitro HLE inhibitor screening model with high sensitivity, accuracy, and repeatability, which provides a better platform for HLE inhibitor screening.