Physicochemical characterization of polyamidoamine (PAMAM) dendrimers of generation 4.0 amine-terminated (DG4.0) and 4.5 carboxy-ended (DG4.5) was done. We have measured the pKa of the inner tertiary amine, the surface primary-amine, and carboxyl-terminal groups. We have conducted UV-Vis absorption and fluorescence emission experiments as a function of pH. We have made a 4th derivative analysis of the UV-Vis absorption experiments and compare the results with classical amide such as dimethylformamide. Our results have permitted us to calculate the pKa of the groups sensible to the pH in both dendrimers and compare the results with theoretical studies. On the one hand, the pKa values found for DG4.0 were 10.0 � 0.5 and 7.1 � 0.2, for primary and tertiary amines groups, respectively. On the other hand, the pKa values found for the DG4.5 were 3.5 � 0.7 and 6.8 � 0.4, for the carboxylic and tertiary amine groups. Our experimental results agree with theoretical ones. We offer probes that the dendrimers have two non-traditional fluorophores, where one of them is the inner amide bond. The dendrimers show fluorescence emission in all the tested pH.