Abstract Simultaneous determination of the organophosphorus pesticides dimethoate, fenthion, diazinon and chlorpyrifos in human blood by HPLC–tandem mass spectrometry was developed and validated. The pesticides were extracted by a simple one-step protein precipitation procedure. Chromatography was performed on a Luna C 18 (30 mm × 2.0 mm, 3 μm) column, using a step-gradient at a flow rate of 0.4 ml/min. The assay was linear from 0.5 to 100 ng/ml ( r 2 > 0.992, n = 24) for all pesticides. The inter- and intra-day accuracy and precision for the method was 96.6–106.1% and <10%, respectively. The lower limit of quantification was 0.5 ng/ml. In conclusion, the method described displays analytical performance characteristics that are suitable for the quantification of these pesticides in cases of acute poisoning.