The determination of the degree of deacetylation of highly deacetylated chitosan by infrared (IR) spectroscopy was significantly improved with the use of partial least squares (PLS). The IR spectral region from 1500 to 1800 cm(-1) was taken as the dataset. Different PLS models resulting from various data pre-treatments were evaluated and compared. The PLS model that gave excellent internal and external validation performance came from the data that were corrected for the baseline and that was normalized relative to the maximum corrected absorbance. Analysis of the PLS loadings plot showed that the important variables in the spectral region came from the absorption maxima related to the amide bands at 1660 and 1550 cm(-1) and amine band at 1600 cm(-1). IR-PLS results were comparable to the results obtained by potentiometric titration. IR-PLS results were found to be more precise and rugged compared to the usual IR absorbance ratio method. This is consistent with the fact that IR spectral resolution is not really high and that the absorption at a single wavelength is influenced by other factors like hydrogen bonding and the presence of water.