Headspace-solid phase microextraction (HS-SPME) was studied and optimised for the determination of four common organophosphorus pesticides (OPPs) in biological samples. Various parameters controlling SPME were studied: choice of SPME fiber, type and content of salt added, preheating and extraction time, desorption time, extraction temperature. Capillary gas chromatographic analysis with nitrogen phosphorus detection (GC-NPD) facilitates sensitive and selective detection of the OPPs: malathion, parathion, methyl parathion and diazinon. Fenitrothion was used as the internal standard. The method was applied to the determination of the pesticides in human biological specimens: whole blood, blood plasma, urine, cerebrospinal fluid, liver and kidney. Limits of detection ranged from 2 to 55 ng/ml depending on pesticide and type of specimen. The developed methodology overcomes limitations and obstacles of conventional methods such as the use of organic solvents, the formation of emulsions and the tedious-cumbersome procedures. The proposed protocol is seen as an attractive alternative to be used in routine toxicological analysis.