Ailanthus altissima (AA) has been used in various anticancer prescriptions and showed excellent therapeutic effect. However, there is no report on the method of quality control and the anti-glioblastoma activity. In this study, we used a combinative method approach consisting of chromatographic fingerprinting and quantitative methods to analyze quality of different samples. The anti-glioblastoma activity and the possible mechanisms were studied by pharmacological methods. The samples were separated on a Kromasil 100-5 C18 column and the flow rate was 0.8 ml/min at 25˚C. The mobile phase was composed of 1% formic acid and 1% methanol-water at a flow rate of 0.8 ml/min. The method was validated and applied to the quantification of different samples. Ten batches of AA from different provinces in China were detected, and we found that the contents of Ailanthone (AT) ranged from 0.21-1.78 mg/g and the relative retention times were similar in different origins. Ten batches of AA were analyzed by the high-performance liquid chromatography (HPLC) fingerprinting method and 19 common peaks were detected. The similarity of 10 batches is <1.5%. The peak areas in different samples were significantly different (0.682-0.954). We also found that AA induced oxidative stress first in U87 cells, then induced ER stress, finally activated the caspases which caused cell apoptosis. In conclusion, a method combining chromatographic fingerprinting and quantitative analysis can be used to control the quality of AA. AA could be used as a medicine or a constituent part of herb prescription to treat glioblastoma.