Composition, level, and arrangement of the structural polysaccharides determine biophysical properties of fungal cell walls. A small amount of a beta(1-->4) linear homopolymer of GlcNAc in the cell wall forms chitin. To study the components of the cell walls and to estimate the quantity of chitin for different strains, two spectroscopic methods were applied. Because chemical and enzymatic methods are destructive, long, and complex, fluorescence and infrared (IR) spectroscopies were applied on cell walls and on chitin enriched fractions. The results were compared to chemical assays. IR spectra allow identifying the structural types of polysaccharides in yeast walls. Fluorescence spectroscopy was not appropriated for a full and accurate quantitative determination of the polymers but revealed level variations similar to results obtained by chemical analytical methods. The infrared spectra, using a chemometric approach (PLS1), allowed a fairly good estimation of chitin in enriched fractions with respect to the chemical assays.