This paper reports a novel approach to screening multi-class pesticides by ion mobility time-of-flight mass spectrometry detection. Nitrogen was selected as mobility gas. After optimization of the different ion mobility parameters, determination of matrix effect on the drift times was conducted using different matrix extracts. The results showed that drift time values are not influenced by the matrix and also are independent of the concentration within the working range for 100 pesticides tested, making drift time a powerful additional identification tool. Based on statistics, 2% variation criteria provides a good fit for all the pesticides targeted, and could be considered as a maximum acceptable criteria associated with the drift time parameter for identification purpose. This 2% value is in agreement with already reported criteria, for instance, for GC or LC retention time in European documents. Finally, the well-known feature of mobility to separate complex mixtures was also tested to obtain purified extracted mass spectra of pesticides present in fruit extract.