Based on proteomics theory， the method for the determination of adulterated donkey meat by liquid chromatography tandem mass spectrometry（LC-MS/MS） was established. In this study， data was acquired by high-resolution mass， and non-labeled quantitative analysis was carried out by proteomics software. The screened data by setting species-specific parameters was analyzed by principal component analysis （PCA） and the accuracy of the model was verified by experiments. Through further data screening， parameters setting and thermal stability investigation， three specific peptides of pig， donkey and horse were selected respectively and then transformed into specific ion pairs to establish LC-MS/MS method. The method could accurately measure the adulteration ratio of 0.5%. The results showed that the method is simple， accurate and reliable.