A sensitive high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method was established for the determination of eplerenone (EP) in human plasma.  The plasma samples of EP were extracted with ethyl acetate and separated by HPLC on a reversed phase C₁₈ column with a mobile phase of 10 mmol^.L⁻¹ ammonium acetate water solution-methanol (30:70, v/v).  EP was determined with electrospray ionization-mass spectrometry (ESI-MS) in the selected ion monitoring (SIM) mode.  The calibration curves were linear over the range of 2 − 4 000 ng^.mL⁻¹ for EP.  The lower limit of quantification was 2 ng^.mL⁻¹.  The method has been successfully applied in the pharmacokinetic study of the EP tablets.  The main pharmacokinetic parameters of EP after oral administration of 25 mg, 50 mg, 100 mg were as follows, t_1/2: (4.9 ± 2.1), (4.7 ± 1.5), (5.9 ± 1.2) h; AUC_0−∞: (4 402 ± 1 735), (8 150 ± 2 509), (13 783 ± 4 102) µg^.h^.L⁻¹; and MRT: (6.2 ± 2.1), (6.6 ± 1.3), and (7.2 ± 1.6) h.  Parameters of EP after oral administration of multiple doses of 50 mg were as follows, t_1/2: (6.1 ± 1.7) h; AUC_ss: (10 071 ± 4 220) µg^.h^.L⁻¹; MRT: (8.1 ± 2.3) h; and DF: (3.2 ± 1.0).