Abstract: High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) were applied to analyze the chemical constituents from acetate, n-BuOH and 95% ethanol extracts of Radix Isatidis. The separation was performed on a Kromasil C18 column (4.6 mm×250 mm×5 μm) with gradient of 0.15% acetic acid/water and methanol as mobile phase with a flow rate of 0.8 mL/min at 25 ℃. The UV spectra of peaks were obtained by a diode array detector (DAD) under the detection wavelength at 254 nm and 265 nm. In order to get exact data of MS and MS² spectra, the atmosphere pressure chemical ionization ion source (APCI) and electrospray ionization ion source (ESI) in positive and negative scan mode were utilized. Up to date, twenty-eight compounds are identified from the crude extracts, including indoles, quinazolinones, organic acids, nucleosides, purines, amino acids, flavones and carbohydrates. Meanwhile, the antioxidant activity of the extracts from Radix Isatidis were estimated by the ferric reducing antioxidant power (FRAP) and radical scavenging (DPPH) methods. The results of the antioxidant activities indicated that the compositions of antioxidant activity are mainly existed in the small polarity part. Indoles, quinazolinones, organic acids and porphyrins are the main antioxidative constituents of Radix Isatidis. This method is rapid and sensitive, which can be used for analyzing the chemical compositions and antioxidant activities of traditional Chinese medicine in the future.