Although experimental design is a powerful tool, it is rarely used for the development of analytical methods for the determination of organic contaminants in the environment. When investigated factors are interdependent, this methodology allows studying efficiently not only their effects on the response, but also the effects of their interactions. A complete and didactic chemometric study is described herein for the optimization of an analytical method involving stir bar sorptive extraction followed by thermal desorption coupled with gas chromatography and tandem mass spectrometry (SBSE-TD-GC-MS/MS) for the rapid quantification of several pesticides in freshwaters. We studied, under controlled conditions, the effects of thermal desorption parameters and the effects of their interactions on the desorption efficiency. The desorption time, temperature, flow, and the injector temperature were optimized through a screening design and a Box Behnken design. The two sequential designs allowed establishing an optimum set of conditions for maximum response. Then, we present the comprehensive validation and the determination of measurement uncertainty of the optimized method. Limits of quantification determined in different natural waters were in the range of 2.5 ng L-1 to 50 ng L-1 and recoveries were between 90 and 104%, depending on the pesticide. The whole method uncertainty, assessed at 3 concentration levels under intra-laboratory reproducibility conditions, was below 25% for all tested pesticides. Hence, we optimized and validated a robust analytical method to quantify the target pesticides at low concentration levels in freshwater samples, with a simple, fast, and solventless desorption step.