We describe here a modified protocol for the simultaneous quantification of specific eicosanoids formed during stimulation of human platelets in vitro with adenosine diphosphate. The eicosanoids thromboxane B 2 (TXB 2 ), arachidonic acid (AA), 12-R-hydroxyeicosatetraenoic acid (12-R-HETE), 12-S-hydroxyheptadecatrienoic acid (12-S-HHTrE) and the internal standard prostaglandin B 1 (PGB 1 ) were extracted from human platelets by liquid-liquid extraction using ethyl acetate. This was followed by derivatization and fluorescent detection prior to analysis by reversed phase liquid chromatography. The highperformance liquid chromatographic method consisted of ODS reversed-phase column (3 µm) and a mobile phase of acetonitrilewater (85:15). TXB 2 and AA plasma calibration curves were linear between 6.25 and 125 ng mL -1 (r 2 > 0.997), whereas for 12-R-HETE and 12-S-HHTrE the curves were linear between 5.0 and 40 ng mL -1 (r 2 > 0.998). All calibration curve standards had <15% CV (coefficient of variation) and between-run precision, and the percentage relative deviation for replicate (n = 6) quality controls was less than 5.5%. The method was adapted to allow the screening of drugs that may affect either one or both of the lipoxygenase and cyclo-oxygenase pathways.