A sensitive and reliable reversed-phase liquid chromatography (RP-LC) with ultraviolet (UV) detection has been developed and validated for the quantification of Icariside II in rat plasma and tissues using Fermononetin as the internal standard. Protein precipitation and liquid–liquid extraction were utilized for plasma and tissue sample preparation, respectively. The analysis was successfully carried out on an Agilent SB-C₁₈ column (5 μm, 4.6 × 250 mm) with the implementation of the following conditions: a mobile phase of phosphoric acid solution (0.1%, v/w)–Acetonitrile (55:45, v/v), a flow rate of 1 mL min⁻¹, a column temperature of 25 °C and a detection wavelength of 270 nm. Good linear relationships of calibration curves were obtained (r ² > 0.9906) over the investigated concentration range with plasma and tissue samples. The lower limit of quantification (LLOQ) and the limit of detection (LOD) were 0.1 and 0.02 μg g⁻¹, respectively (for plasma sample, they were 0.05 and 0.1 μg mL⁻¹, respectively). The developed method which was embodied with good precision, accuracy, recovery and stability was corroborated to satisfy the requirements for biomedical sample analysis. This method has been successfully applied to tissue distribution study of Icariside II in rats after a single intravenous dose at 12.5 mg kg⁻¹. Results suggested that Icariside II was distributed to rat tissues rapidly with greater initial concentrations in kidney, lung and liver. Moderate initial distributions were obtained in rat muscle, heart, bone, spleen and plasma. Low amount of Icariside II was detected in testes, and no Icariside II could be detected in the brain.