Poly(amido amine)s (PAAs) are a family of water soluble polymers endowed with properties which find useful applications in the medical field. In order to perform degradation studies on PAAs by using GPC in aqueous media, a calibration curve was derived by following a purposely developed procedure. A set of PAA samples having different molecular weights were synthesized, by means of stepwise polyaddition reactions, carried out with nonstoichiometric ratios of the monomers. A careful characterization of the samples was performed by means of H-1, C-13, and bidimensional homo- and heterocorrelated NMR spectrometry. Particular attention was devoted to the quantitative determination of end functionalities, which enabled us to calculate the number-average molecular weight of PAA samples. The GPC results, obtained in buffered aqueous solution, were combined with NMR data on number-average molecular weight, and by means of a nonlinear least squares fitting procedure, following iterative routines, a linear calibration curve of log M versus retention times was obtained. Weight averages and the polydispersity index for the PAA samples were then determined. Finally, from viscometric measurements,the Mark-Houwink constants were also determined using number-average molecular weights.