The purpose of the present study was the development of an accurate method to determine the degree of substitution (DS) of modified hyaluronic acid (HA) by proton nuclear magnetic resonance (1H NMR) spectroscopy. The influence of the effect of ionic strength and pH on 1H NMR spectra of HA was studied. The results showed a correlation between the conformation of HA in solution and the quality of the 1H NMR spectra. The best spectra with full proton mobility are obtained when HA is dissolved in D2O with 2 M NaCl or D2O with 0.1 M NaOD with a maximum concentration of 5 mg/ml. Under those conditions the size of the polymer coils is reduced below the concentration of chain overlap point, all the protons show the same response and a correct degree of substitution can be determined.