Analysis of pesticides and their metabolites, especially in different matrices, continues to be an active research area closely related to food safety and environmental issues. Despite the fact that the use of pesticides in agricultural practice has resulted in a substantial increase in crop yield, pesticides and their metabolites can affect human health, pollute the natural environment, and disturb the equilibrium of the ecosystem. Their toxic effects are compound-specific and include several mechanisms of action. Pesticides comprise a large number of substances that belong to completely different chemical groups. The nature of the samples necessitates the use of preconcentration, cleanup, and separation techniques. Therefore, various extraction methods are suited for matrix separation and analyte preconcentration, followed by cleanup and chromatographic procedures with various detection techniques and their combinations for the actual determination.