Thromboxane B2, prostaglandin E2 and leukotriene B4 were simultaneously extracted from human whole blood without any time‐consuming evaporation or derivatization process, and directly determined by liquid chromatography/ionspray mass spectrometry. In order to obtain high sensitivity and specificity, the chromatographic and mass spectrometric conditions were optimized. Use of the turbo ionspray method plus a microbore column gave excellent sensitivity. A special column‐switching technique allowed the injection of large sample volumes with minimum band broadening. The calibration graphs were linear from a few tens of pg ml−1 to 10 ng ml−1. This simple and rapid method was validated using human whole blood samples to which known amounts of standards had been added. Because the levels of these mediators in biological samples are elevated in some diseases or owing to stimulants, this method is very useful for determining them in such cases.