alpha,beta-poly[(N-hydroxyethyl)-DL-aspartamide] (PHEA) is a new synthetic polymer which is of interest in biomedical applications. In this paper, the molecular characterization of PHEA by multi-angle laser light scattering and viscometry off-line and on-line to a size exclusion chromatography system is reported. These techniques furnish an exhaustive and consistent characterization of the PHEA polymer. The fractionation of the PHEA macromolecules was relatively simple. Using an aqueous mobile phase of medium ionic strength, the elution was substantially regular and the macromolecules were not aggregate. The molar mass M of four PHEA samples approximately ranges from 46 to 53 K g/mol, the intrinsic viscosity [eta] ranges from 0.22 to 0.26 dl/g and the gyration radius R-g is also estimated. Both the slopes of the [eta] = f(M) and R(g)f(M) power laws prove that PHEA macromolecules in aqueous solution are flexible coils. Besides, the second virial coefficient value proves that the used mobile phase is a good solvent for PHEA. The molecular characterization of PHEA was also performed through conventional SEC and universal calibration and the estimated molar mass was substantially higher than that evaluated by absolute methods.