The purpose of this article is to provide an overview of the main characteristics of organophosphorus pesticides (OPPs), such as their physicochemical characteristics, sources, fate and distribution in the environment. It covers regulatory compliance from the Commission of the European Communities (CEC) and the United States National Pesticide Survey (USNPS), regarding the maximum permissible levels of the most toxic and persistent OPPs and their transformation products (TPs) in water. This article also describes the analytical methods currently used to monitor pesticides and their degradation products in water and food matrices. A major part concerns sample preparation techniques and includes traditional liquid–liquid extraction (LLE) and novel techniques based in automated solid‐phase extraction (SPE), either on‐line or off‐line. New sorbent materials, their applications and the parameters involved in the extraction are described. Chromatographic methods used for their determination are discussed in detail. Much emphasis has been given to gas chromatography (GC) and liquid chromatography (LC) coupled to mass spectrometry (MS) as a confirmation technique and mass spectral information on several pesticides has been included. The final section is devoted to method validation, with the aim of providing a basis and a framework of information for quality assurance (QA) and interlaboratory calibration studies.