Abstract: A new concept for sample cleanup, based on size exclusion of macro molecular compounds in matrix, has been developed by coupling of gel permation chromatography (GPC) with LC-MS/MS. Heart-cutting two-dimensional liquid chromatography tandem mass spectrometry system was used by transferring the target elution through a loop. In the two dimensional liquid chromatography -tandem mass spectrometry system, a GPC column was employed in the first dimension to separate the macromolecule interference compound weight. After split, the target fluent were heart-cutting to a loop connected to a 6-way valve. Then the valve switched, so the second dimensional mobile phase took the target compounds for further reversed phase liquid chromatographic separation. Finally, about 80 different species of pesticide residues were determined qualitatively and quantitatively under the multi reaction monitoring (MRM) mode. The recoveries of most pesticides were in the range of 72%-136% and the limits of quantitation (LODs) for this method were from 0.022 to 0.152 mg/kg. Compared to conventional approaches for pesticide residue analysis in tobacco, the new technique involves less contamination of ion source in MS without sacrificing method LODs. In addition, the 2DLC-MS/MS method was fully automatic and the results were also accurate when utilized to detection of tobacco samples.