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Objective: Pretreatment of biological samples is the most critical step in pharmacokinetic studies, especially pre-treatment of plasma samples. The pretreatment of biological samples in pharmacokinetic study of Traditional Chinese Medicines (TCM) is difficult due to the complexity of the ingredients. An organic solvent system ethyl acetate: acetone (10: 1) solution used for liquid-liquid extraction has been developed in this study and compared with the commonly used protein precipitation method. Methods: Rats, Beagle dogs and humans plasma samples were adopted in this study in order to demonstrate the universality of the pretreatment method. Feasibility of this pretreatment method was also verified through its application to the pharmacokinetics of rhubarb in rats. Results: According to the results of extraction recovery matrix effect, it was indicated that the liquid-liquid extraction methods with new organic solvent might be suitable for variety of structures of compounds and various types of plasma samples. The pharmacokinetic study result showed that the developed pretreatment method could successfully be used for simultaneous determination of three active compounds modin, emodin-8-O-β-D-glucopyranoside (EDG) and rhein in rat plasma with high sensitivity, accuracy, and recovery by liquid chromatography tandem mass spectrometry (LC-MS/MS). Conclusion: The pretreatment method of liquid-liquid extraction methods with new organic solvent could be successfully applied for multi-component pharmacokinetics of TCM. Keywords: Pretreatment method, Liquid-liquid extraction, Protein precipitation, LC-MS/MS, Rhubarb