The basic premise of gel permeation, molecular sieving, or size exclusion chromatography is based on the difference in different molecular weights or size and is one effective method to isolate and analyze bio-macromolecular substances. Gel permeation is also called size exclusion chromatography, which uses porous material as stationary phase of the size of 50–3000 angstroms which is akin to molecular size. Small molecules pass rapidly than large molecules, and separation becomes possible. Complete description of experimental layout and different stationary phases with different mobile phases has been given in this chapter. The more widely used extraction techniques for pesticides are Soxhlet extraction (SOX), shake-flask, liquid–liquid extraction (LLE), solid-phase extraction (SPE), solid-phase micro-extraction (SPME), matrix solid-phase dispersion (MSPD), supercritical fluid extraction (SFE), accelerated solvent extraction (ASE), and microwave-assisted extraction (MAE). ASE is an extraction technique that speeds up the extraction process and reduces the total amount of solvent used. Special reference has been emphasized on pesticide residue analysis in the backdrop of gel permeation chromatography.