Abstract: AIM: To develop a sensitive and accurate analytical method for the profiling, identification and simultaneous quantification of the chemical constituents in Aurantii Fructus Immaturus extracts. METHODS: A high performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-MS/MS) method was established for the characterization and quantification of multi-constituents in Aurantii Fructus Immaturus extracts. Chromatographic separation was achieved on a C18 column using a detailed gradient program with 2% formic acid and acetonitrile as the mobile phase. Mass spectrometry was in the negative mode for identification, and a diode array detector was set at 283 nm for quantification. RESULTS: Eighteen compounds were identified, or tentatively identified, by comparing their retention times, UV and MS spectra with those of authentic compounds or literature data, including 12 flavonoids, 5 coumarins and a limonoid in Aurantii Fructus Immaturus extracts. Twelve of the major compounds were determined simultaneously by HPLC-DAD. The fragmentation pathways of the authentic references were also investigated for the structural identification of the constituents in the extracts. There have been no reports on the simultaneous and comprehensive analysis of both the major and minor compounds in Aurantii Fructus Immaturus extracts. CONCLUSIONS: The method validation indicated that the developed analysis method could be employed as a rapid, effective technique for the structural characterization and simultaneous quantification of multi-constituents in this botanical extract. This work is expected to provide comprehensive information for the quality evaluation of Aurantii Fructus Immaturus extracts, which should be a valuable support for the further study and development of this raw material and its related medicinal products.