Abstract: The concoction process is a vital technique for reducing the toxicity and enhancing the efficacy of Chinese herbal medicines. To examine the alterations in the chemical compositions of Aurantii Fructus before and after stir-frying in diverse origins, a method of internal extraction electrospray ionization mass spectrometry (iEESI-MS) was developed. It combines the advantages of internal extraction and electrospray ionization mass spectrometry, and is capable of detecting target molecules in complex samples with highly efficient and sensitive manner. iEESI-MS was employed to directly analyze the Aurantii Fructus samples before and after processing without complex sample pretreatment, which can obtain their fingerprints and identify the principal difference substances present in the samples before and after processing. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used for data handling. The aforementioned screened main differences in substances were subjected to analysis in a variety of origins. The experimental conditions were optimized including the temperature of ion transport tube, spray voltage, flow rate of the extractant, proportion of methanol spray solution and distance from the spray port to the mass spectrometry port, etc. The results showed that a total of 35 substances are identified in the Aurantii Fructus samples, including flavonoids, coumarins, alkaloids, volatile oils, and amino acids. The chemical composition of Aurantii Fructus exhibites notable differences before and after preparation. Flavonoids constitute approximately two-thirds of the identified distinct compounds, and serve as crucial quality markers for differentiating the origin and quality of Aurantii Fructus. The results of the human heat map analysis of Aurantii Fructus from disparate origins in Hunan, Sichuan and Jiangxi demonstrated that the variation of the 12 distinct substances present in Aurantii Fructus from different origins is considerable. This indicated that the contents of these 12 substances are inconsistent among the different origins, which is related to the disparate quality of Aurantii Fructus from different origins in terms of its medicinal effects. This study provides a novel approach to ambient mass spectrometry for the quality identification of herbs such as Aurantii Fructus and the material basis of efficacy change before and after processing.