Pueraria is a traditional Chinese medicine and has been used as a food and medicine dual purposes herb for 2000 years. The dried root of it includes Pueraria lobata and Pueraria thomsonii, which is prescribed for the treatment of fever, acute dysentery, diarrhoea, thirst, diabetes and hypertension. However, the root of Pueraria is liable to accumulate contamination such as pesticide residues and heavy metals. Therefore, it is necessary to develop a series of quality control methods of Pueraria for analysis of its active and toxic compounds. Firstly, the microwave-assisted extraction was used as a rapid preparation method for extraction of isoflavones in Pueraria. The ultra-high performance liquid chromatography with sub-2 μm column and regular high performance liquid chromatography with new shell-type column were performed for fast separation of isoflavones. In order to acquire comprehensive signals, different detectors including ultraviolet detector, triple quadrupole mass spectrometer and time-of-flight mass spectrometer were utilized for the qualitative and quantitative analysis of 13 isoflavones in Pueraria. Secondly, a fast gas chromatography coupled with mass spectrometry using large volume injection with programmed temperature vaporizer was developed for the simultaneous determination of multiple pesticide residues in Pueraria. The different sample preparation methods including QuEChERS method, solid phase extraction, solid phase microextraction, pressurized liquid extraction coupled with gel permeation chromatography and selective pressurized liquid extraction were compared. Results indicated that selective pressurized liquid extraction and GC-MS/MS was a sensitive and reliable analytical system for monitoring pesticide residues in Pueraria. Lastly, an inductively coupled plasma mass spectrometry was employed for determination of 10 heavy metals in Pueraria. Meanwhile, an ionic chromatography coupled with inductively coupled plasma mass spectrometer was performed to analyze the chromium species including trivalence chromium and hexavalence chromium. Results indicated that trivalence chromium was the major species in Pueraria. In summary, all analytical methods were rapid, sensitive, selective and reliable. Those validated methods were helpful to establish and improve the quality control system of Pueraria. Meantime, those quality control methods were helpful to evaluate the active and toxic substances in Pueraria.