A　pre-column　derivatization　method　was　developed　to　analyze　polyamidoamine　(PAMAM)　dendrimers.　The　fluorescein　isothiocyanate　(FITC)　derivation　method　improved　the　retention　of　PAMAM　dendrimers　on　the　reversed-phase　column　of　high　performance　liquid　chromatography　(RP-HPLC).　The　derivation　benefited　the　UV-Vis　detection　of　PAMAM,　although　the　fluorescence　of　FITC　was　quenched　by　PAMAM　dendrimers.　The　primary　amine-terminated　PAMAM　dendrimers,　generation　3　(PAMAM-NH2　(G3)),　were　quantitatively　determined　by　UV-Vis　spectroscopy　over　the　concentration　range　of　10-80　μg　mL-1,　with　the　calibration　curve　expressed　as　A　=　131.6C　(μg　mL-1)　-　295.6　(R2　=　0.9901)　and　the　detection　limit　of　2.7　μg　mL-1.　The　binding　ratio　of　FITC　to　PAMAM-NH2　(G3)　was　determined　as　8:1　by　liquid　chromatography-electrospray　ionization-ion　trap　mass　spectrometry　(LC-ESI-IT-MS).　©　The　Royal　Society　of　Chemistry　2014.