Abstract: The objective of this study is to develop an in vitro screening method for nasal absorption of insulin. First, the adaptability of in situ rat nasal perfusion test for the study of insulin was investigated. It was found that insulin was liable to be absorbed on the silicone tube and the traditional method is not suitable. However, addition of 0.001% Labrasol into the perfusate can effectively solve this problem. A modified method suitable for in situ rat nasal perfusion of insulin was established with the addition of 0.001% Labrasol into the perfusate. Using the modified method, effect of pH and drug concentration on the absorption of insulin in the nasal cavity was further investigated. The results suggest that compared with pH 4.5 and pH 7.4, the drug absorption rate was the lowest at pH 6.0. The intranasal absorption mechanism of insulin may be passive diffusion.