A highly rapid and sensitive liquid chromatographic–electrospray ionization tandem mass spectrometric(LC–ESI-MS/MS) method was developed and validated for the determination of trans-δ-viniferin(Rs-1) in rat plasma, urine and feces. All biological samples were prepared by liquid–liquid extraction and hesperetin was included as an internal standard(IS). Chromatographic separation was achieved on a shim-pack XR-ODS column using a gradient mobile phase. MS/MS detection was performed by negative ion electrospray ionization. The method was sensitive with a lower limit of quantification of 1.42 ng/m L and linear over the range of 1.42–2172 ng/m L in all matrices. The method was applied to study the pharmacokinetics, bioavailability, metabolism, and excretion of Rs-1 in rats following a single oral or intravenous dose. Two metabolites, Rs-1 glucuronide and Rs-1 sulfate, were detected in plasma and in urine after administration of Rs-1. The absolute oral bioavailability of Rs-1 was2.3%, and the total absorption rose to 31.5% with addition of its glucuronide and sulfate metabolites. Only0.09% of the gavaged dose, including Rs-1 and metabolites, was excreted in the urine, while 60.3% was found in the feces in unchanged form. The results indicate that both poor absorption and extensive metabolism were the important factors that led to the poor bioavailability of Rs-1, which can provide a basis for further studies on structural modification and dosage form design. A highly rapid and sensitive liquid chromatographic-electrospray ionization tandem mass spectrometric (LC-ESI-MS / MS) method was developed and validated for the determination of trans-δ-viniferin (Rs-1) in rat plasma, urine and feces. All biological samples were prepared by liquid-liquid extraction and hesperetin was included as an internal standard (IS). Chromatographic separation was achieved on a shim-pack XR-ODS column using a gradient mobile phase. MS / MS detection was performed by negative ion electrospray The method was sensitive with a lower limit of quantification of 1.42 ng / m L and linear over the range of 1.42-2172 ng / m L in all matrices. The method was applied to study the pharmacokinetics, bioavailability, metabolism, and excretion of Rs-1 in rats following a single oral or intravenous dose. Two metabolites, Rs-1 glucuronide and Rs-1 sulfate, were detected in plasma and in urine after administration of Rs-1. The absolute oral bioavailability of Rs-1 was2 .3% , and the total absorption rose to 31.5% with addition of its glucuronide and sulfate metabolites. Only 0.09% of the gavaged dose, including Rs-1 and metabolites, was excreted in the urine, while 60.3% was found in the feces in unchanged form. The results indicate that both poor absorption and extensive metabolism were the important factors that led to the poor bioavailability of Rs-1, which can provide a basis for further studies on structural modification and dosage form design.