Imidol hydrochloride is a novel drug for the treatment of hepatitis B virus infection. A simple, special and sensitive solid-phase extraction liquid chromatography–tandem mass spectrometry method for determination of imidol in human plasma and urine was developed for the first time and applied to a pharmacokinetic study. The chromatographic separation was achieved on a C₁₈ column (50 x 2.1 mm, 3.5 μm) using gradient elution with acetonitrile and water both containing 0.1% acetic acid at a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via a positive eletrospray ionization source. The mass transition pairs of m/z 517.8 --> 325 and m/z 298 --> 174 were used to detect imidol and the (-)-clausenamide (internal standard), respectively. The retention times of imidol and (-)-clausenamide were 2.5 and 2.7 min, respectively. Linearity, accuracy, precision, recovery, matrix effect, dilution test and stability were evaluated during method validation over the range of 0.2-500 ng/mL in human plasma and 0.5-500 ng/mL in urine. The method was successfully applied to a clinical pharmacokinetic study of imidol in healthy volunteers following oral administration.