This study aims to develop a standard protocol for the relative bioavailability testing of doxazosin mesylate tablets. For this purpose, a simple rapid and selective LC–MS method using a single quadrupole mass spectrometer was developed and validated to determine the concentration of doxazosin mesylate in human plasma. Using this method, we carried out a study of relative bioavailability. N-Hexylane-tertiary butyl methyl ether (1:1, v/v) was used to extract doxazosin mesylate and terazosin (internal standard, I.S.) from an alkaline plasma sample. LC separation was performed on a Thermo Hypersil-Hypurity C18 (5 μm, 150 mm × 2.1 mm) using aqueous solution (20 mmol/l ammonium acetate, pH 4.28), methanol and acetonitrile (55:10:35, v/v/v) as the mobile phase. The retention time of doxazosin and the internal standard was 2.7 and 1.8 min, respectively. Quadrupole MS detection was done by monitoring at m/z 388 (M + 1) corresponding to doxazosin mesylate and at m/z 452 (M + 1) for I.S. The assay method described above showed acceptable precision, accuracy, linearity, stability, and specificity. The bioavailability of doxazosin mesylate was evaluated in 12 healthy Chinese male volunteers. The following pharmacokinetic parameters were elucidated after administering a single dose of 4 mg doxazosin. The area under the plasma concentration versus time curve from time 0 to 72 h (AUC0–72 h) 743.4 ± 149.5 ng h/ml; peak plasma concentration (Cmax) 47.66 ng/ml; time to Cmax (Tmax) 3.0 ± 1.0 h; and elimination half-life (t1/2) 18–20 h. The method was successfully used to determine the relative bioavailability of doxazosin mesylate.