Saffron (stigmas of Crocus sativus L.) is a well-known spice with many attributed therapeutic uses throughout centuries. Although studies have demonstrated that crocetin and crocins from saffron have various biological functions, issues concerning the route and way of saffron administration, the absorption and metabolism of saffron carotenoids in humans have not been answered yet. In the present study, an isocratic reversed-phase liquid chromatographic method was developed and validated for the determination of crocetin in plasma. Samples were pre-treated by solid phase extraction (recoveries >72%) and were chromatographed on a Luna C-18 column (4.6mm×250mm, 5μm) with a mobile phase consisting of methanol-water-trifluoroacetic acid (75.0:24.5:0.5, v/v/v) at a flow rate of 1.0mLmin(-1). The HPLC method developed resulted in sharp peaks at 10.7 (trans-crocetin) and 18.6min (cis-crocetin), whereas the calibration curve of total crocetin in plasma displayed a good linearity for concentrations of 0.020-20μM (R(2)=0.999). Specificity, precision, accuracy and stability were studied with spiked plasma samples and were acceptable. The developed method was applied to the determination of crocetin levels in plasma of four healthy human volunteers before and after consumption of one cup of saffron tea (200mg of saffron in 80°C water for 5min). Results showed that the concentration of crocetin was high after 2h (1.24-3.67μM) and still determined after 24h (0.10-0.24). Interestingly, the percentage of the cis-isomer ranges from 25 to 50%, suggesting in vivo isomerization.