Abstract: High performance liquid chromatography-atmospheric pressure chemical ionization/mass spectrometry (HPLC-APCI/MS) and electrospray ionization tandem mass spectrometry (ESI-MS²) were applied to analyze the chemical constituents from acetate, n-BuOH and 95% ethanol extracts of Isatidis Folium. The separation was performed on a Kromasil C18 column (4.6 mm×250 mm×5 μm) with the gradient of 0.15% acetic acid/water-methanol as mobile phase with a flow rate of 0.8 mL/min at 25 ℃. The UV spectra 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 both positive and negative mode were utilized. Up to date, thirty-nine different compounds were identified from the crude extracts, including alkaloids, organic acids, nucleosides and purines, amino acids, flavones, carbohydrates, porphyrin and epigoitrin. Meanwhile, the antioxidant activity of the extracts from Radix Isatidis were successfully 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 were mainly existed in the small polarity part. Indoles, quinazolinones, organic acids and porphyrins might be the main antioxidative constituents of Radix Isatidis. This method is rapid and sensitive that can be used for analyzing the chemical compositions and antioxidant activities of traditional Chinese medicine.