Homoeriodictyol-7-O-β-D-glucopyranoside (HEDT-Glu) was isolated from Viscum coloratum and identified by MS, ¹H- and ¹³C-NMR. A HPLC method was developed for determination of HEDT-Glu in rat plasma and tissues. All biological samples were pretreated by protein precipitation with acetone. Vanillin was selected as internal standard. The mobile phase consisted of methanol–water–glacial acetic acid (45 : 55 : 0.5, v/v/v). Good linearity were observed over the concentration ranges of 0.1—200.0 μg·ml⁻¹ in rat plasma and 0.05—5.0 μg·ml⁻¹ in tissues. Both intra- and inter-day precisions of HEDT-Glu, expressed as the relative standard deviation, were less than 13.1%. Accuracy, expressed as the relative error, ranged from −0.8 to 5.4% in plasma and from −5.6 to 9.4% in tissues. The mean extraction recovery of HEDT-Glu was above 73.17% in biological samples. The described assay method was successfully applied to the pre-clinical pharmacokinetic study of HEDT-Glu. After intravenous administration of HEDT-Glu to rat, AUC and CL_tot were 16.04±3.19 μg·h·ml⁻¹ and 0.85±0.17 l·kg⁻¹·h⁻¹, respectively. T_1/2,α and t_1/2,β were 0.06±0.01 h and 1.27±0.31 h, respectively. HEDT-Glu was cleared from the blood and mainly distributed to the liver and small intestine.