Chondroitin sulfate (CS)and dermatan sulfate (DS) are negatively charged polysaccharides found abundantly in animal tissue and have been extensively described to play key roles in health and disease. The most common method to analyze their structure is by digestion into disaccharides with bacterial chondroitinases, followed by chromatography and/or mass spectrometry. While studying the structure of oncofetal CS, we noted a large variation in the activity and specificity of commercially available chondroitinases. Here studied the kinetics of the enzymes and used high-performance liquid chromatography-mass spectrometry to determine the di- and oligosaccharide products resulting from the digestion of commercially available bovine CS A, shark CS C and porcine DS, focusing on chondroitinases ABC, AC and B from different vendors. Application of a standardized assay setup demonstrated large variations in the enzyme-specific activity compared to the values provided by vendors, large variation in enzyme specific activity of similar enzymes from different vendors and differences in the extent of cleavage of the substrates and the generated products. The high variability of different chondroitinases highlights the importance of testing enzyme activity and monitoring product formation in assessing the content and composition of chondroitin and DSs in cells and tissues.