This study reports on a new approach to a well-known method to reduce the molecular weight of chondroitin sulfate (CS) using a non-specific enzyme. Here, the enzyme hyaluronidase was utilized to depolymerize raw CS (19.2 kDa) resulting in low molecular weight CS (LMWCS) as a product. A simple ultrafiltration process utilizing circular-flat membranes with different MWCO was utilized to recover the fraction of interest (5.6 kDa). LMWCS characterized by GPC/LALLS, viscometric analyses and FTIR and NMR spectroscopies. The enzymatic depolymerization kinetics was investigated and some important parameters but poorly demonstrated in the literature, such as the rate of depolymerization for bond cleavage (0.308 mol/L h); depolymerization rate constant (2.98 x 10(-3)h(-1)); and the relationship between M and Ms, is provided. The present work could be a basis on which to present a suitable and original method to obtain and recover LMWCS. Furthermore, it could be a useful reference work for the investigation of how to improve the biological activity of CS in biomedical and pharmaceutical devices by reducing the CS molecular weight.