An unexpected 1H NMR invisible fraction (IF) for chitosan (CS) and CS-g-PEG is reported. The presence of this IF is remarkable considering that solution NMR is recognized as the method of choice for studying structural modifications in CS, including the degrees of acetylation (DA) and substitution (DS). In spite of IF figures as high as 50%, this IF does not interfere in the correct determination of the DA by 1H NMR, pointing to a homogeneous distribution of acetyl groups along the visible and invisible fractions. Quite in contrast, the IF negatively biases the determination of the DS in CS-g-PEG, with relative errors as high as 150% in a broad range of temperatures, pH values, and concentrations. This fact raises concerns about the accuracy of previously reported DS data for CS-g-PEG and many other CS copolymers. Efficient user-friendly conditions have been developed for the correct determination of the DS of CS-g-PEG by depolymerization by nitrous acid.