Among ecological processes, the decomposition of leaf litter is central to the functioning of lotic ecosystems. Chemical contamination counts as a widespread stressor in freshwater ecosystems. This is especially the case in rivers draining agricultural areas, where the diffuse pollution of pesticides to freshwaters can modulate the leaf litter decomposition processes. Then the determination of pesticide concentration in the different compartments (water, leaf litter) of the aquatic system is required. The analysis of trace of pesticides in complex matrix such as leaves requires efficient analytical techniques to reach appropriate sensitivity and especially specificity (to limit matrix effects). Prior analyzing complex samples, one or several sample preparation step(s) are mandatory to make the sample less complex thereby limiting matrix effects occurring in MS and hence facilitating both identification and quantification of the pesticides. In this work, QuEChERS conditions was optimized using an experimental design. The resulting extract was passed through a SPE cartridge (Solid Phase Extraction) to increase the selectivity of the sample preparation. Before injection into the chromatographic system, a filtration step was carried out to avoid column clogging. Despite this optimized sample preparation, severe matrix effects occurred when one dimensional liquid chromatography was hyphenated to MS-QTOF (RPLC-MS-QTOF) thereby leading to some undetected pesticides. With a view to limit both loss in sensitivity due to sample dilution and total analysis time affected by the numerous sample treatment steps, an on-line comprehensive two dimensional liquid chromatography approach (LC x LC-QTOF) was applied using Hypercarb and C18 columns in the first and the second dimension respectively. In addition to the advantages cited above, it was found that the resulting RPLC x RPLC separation offered much larger peak capacity than the 1D-LC one. This gain in peak capacity allowed to significantly reduce matrix effects in MS and hence to obtain unambiguous identification of pesticides. As a conclusion, LCxLC-QTOF approach seems to be very promising to deal with complex environmental matrices both for targeted and un-targeted objectives.