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To study pharmacokinetic properties of puerarin poly(amido amine) (PAMAM) dendrimer complex, a sensitive liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed and validated to determine puerarin in rabbit aqueous humor using microdialysis sampling. Astilbin was used as the internal standard. The linear range for puerarin was from 2 to 1000 ng/mL (r = 0.9986) based on 20 μL of aqueous humor. The coefficients of variations for intra-day and inter-day precisions were less than 10.0%, and the relative error of accuracy was within ±6.3%. The mean extraction recovery of puerarin varied from 80.4% to 85.5%. Microdialysis provides a complete concentration versus time profile. A significant difference was observed in main pharmacokinetic parameters of Cmax, AUC and t1/2 between puerarin solution and puerarin PAMAM dendrimer complex. Complex formation resulted in an obvious increase in bioavailability of puerarin after topical administration to rabbit according to the above LC-MS/MS assay method.