The review deals with isolation and pretreatment steps in pesticide residue analysis in fruit and vegetable matrices. The basic principles and an overview of practical applications is given for classic solvent and modern instrumental extraction procedures, such as liquid extraction, supercritical fluid extraction, accelerated solvent extraction and solid phase matrix dispersion. The review is also focused on comparison of advantages and limitations of different pretreatment steps such as liquid-liquid re-extractions, solid phase extraction, gel permeation chromatography, solid phase microextraction, stir bar sorptive extraction and direct sample introduction.