The article presents enzymatic methods for the precise and rapid determination of small quantities of isomeric chondroitin sulfates (A, B, and C) in mixtures with other mucopolysaccharides. The methods utilize chondroitinase-ABC, chondroitinase-AC, chondro-4-sulfatase, and chondro-6-sulfatase to catalyze specific reactions that produce measurable products. These methods can be applied to determine chondroitin sulfates in as little as 4 ml of normal urine. The authors describe three analytical procedures (Methods I, II, and III) that measure the release of unsaturated disaccharides, inorganic sulfate, or the formation of ΔDi-OS, respectively. These methods are sensitive, specific, and can handle mixtures containing hyaluronic acid, chondroitin, heparin sulfate, keratosulfate, and heparin. The article also discusses the application of these methods to labeled materials, allowing for the measurement of radioactivity alone, which is particularly useful for detecting small amounts of labeled chondroitin sulfates in tissues or mixtures. The methods are validated through synthetic mixtures and normal human urine, demonstrating their reliability and practicality for clinical and research applications.The article presents enzymatic methods for the precise and rapid determination of small quantities of isomeric chondroitin sulfates (A, B, and C) in mixtures with other mucopolysaccharides. The methods utilize chondroitinase-ABC, chondroitinase-AC, chondro-4-sulfatase, and chondro-6-sulfatase to catalyze specific reactions that produce measurable products. These methods can be applied to determine chondroitin sulfates in as little as 4 ml of normal urine. The authors describe three analytical procedures (Methods I, II, and III) that measure the release of unsaturated disaccharides, inorganic sulfate, or the formation of ΔDi-OS, respectively. These methods are sensitive, specific, and can handle mixtures containing hyaluronic acid, chondroitin, heparin sulfate, keratosulfate, and heparin. The article also discusses the application of these methods to labeled materials, allowing for the measurement of radioactivity alone, which is particularly useful for detecting small amounts of labeled chondroitin sulfates in tissues or mixtures. The methods are validated through synthetic mixtures and normal human urine, demonstrating their reliability and practicality for clinical and research applications.