A simple sensitive and selective liquid chromatographic–tandem mass spectrometric method was developed and validated for the quantification of azithromycin in human plasma. Roxithromycin was used as the internal standard. The chromatographic separation was performed on a SunFire C18, 50 mm × 2.1, 3.5 μm column at 30 °C with mobile phase consisted of 1.54 g ammonium acetate, 250 mL water, 570 mL acetonitrile, 180 mL methanol and 0.6 mL glacial acetic acid. Flow rate was 0.2 mL min⁻¹. The work-up procedure involved a liquid–liquid extraction of the compounds. Mass spectrometric data were acquired in single ion monitoring. MRM mode of the ions 749.58 > 591.6 and 837.64 > 158.2 for azithromycin and roxithromycin, respectively. The method was validated in the concentration range of 2–1,000 ng mL⁻¹. Absolute recovery of azithromycin was 81.97%. Retention time for azithromycin was 0.9 and 1.1 min for roxithromycin. The run time was 2 min. This method was found suitable to analyse human plasma samples for application in pharmacokinetic, pharmacodynamic, bioavailability/bioequivalance studies.