A high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for the determination of mizoribine in human serum   using thiamphenicol as internal standard (IS).  The serum samples of mizoribine were precipitated with acetonitrile and separated by HPLC on a reversed phase C₁₈ column with a mobile phase of 0.1% ammonium acetate water solution-methanol (47∶53, v/v).  Mizoribine and IS were detected in the multiple reaction monitoring mode with precursor/product ion transitions of m/z 258.2/126.0 and 354.1/185.2, respectively.  The calibration curves were linear over the range of 0.02 − 2 μg·mL⁻¹ for mizoribine.  The limit of quantification (LOQ) was     0.02 μg·mL⁻¹ with acceptable precision and accuracy.  The validated method was successfully applied for the evaluation of a bioequivalence study on Chinese healthy volunteers.  The main pharmacokinetics parameters after oral  administration of 100 mg mizoribine test or reference formulation were as follows: C_max (1.00 ± 0.21), (1.00 ± 0.22) μg·mL⁻¹; AUC_0−∞ (6.72 ± 1.39), (6.48 ± 1.44) μg·h·mL⁻¹; t_1/2 (2.77 ± 0.26), (2.66 ± 0.29) h;     t_max (2.95 ± 0.78), (2.84 ± 0.50) h.