A rapid and sensitive method for the quantitative analysis of azithromycin in human tears by LC-MS/MS was developed and validated. Following extraction from collected Schirmer tear strips by methanol-water (4:1, v/v), the analyte and IS (azithromycin-d3) were separated on a Waters Atlantis (TM) dC(18) column (2.1 mm x 30 mm, 3 pm) by gradient elution with 0.1% (v/v) formic acid in methanol-water (1:9) and methanol-acetonitrile (9:1) as the mobile phase. Electrospray ionization in positive ion mode and MRM were used to monitor the ion transitions at m/z 749.6 -> 591.6 (azithromycin) and 752.4 -> 594.4 (azithromycin-d3). The results indicated that the method had excellent sensitivity and specificity. The analyte appeared to have good linearity in the range of 5-1000 ng/mL. Both the intra-batch and inter-batch precisions (in terms of RSD) were <10%, and the accuracies (in terms of RE) were within +/- 15%. The lower limit of quantification, matrix effect, extraction recovery, stability and dilution integrity were also evaluated and satisfied the validation criteria. Artificial tears served as the surrogate matrix, and no matrix difference was found when compared with that of real human tears. Finally, this method was successfully applied in an ocular pharmacokinetic study in healthy volunteers following instillation of azithromycin eyedrops.