Glucosamine hydrochloride and chondroitin sulphate are glycosaminoglycans (GAGs) compound which is a major structural component of bone that form proteoglycans. Both of these compounds can take care of bone health by stimulating the synthesis of synovial fluid and inhibit the degradation of joint cartilage, so it can be used for the treatment of osteoarthritis. The aimed of this study were obtain selective analytical method for the determination of glucosamine hydrochloride and chondroitin sulphate levels in tablet and cream dosage forms. After derivatization using orthophtalaldehyde and 2-mercaptoethanol (OPA/2-ME), samples were analyzed using high performance liquid chromatography (HPLC) with fluorescence detector at excitation wavelength of 335 nm and emission wavelength of 445 nm.. Glucosamine has a primary amine group that can be derivatized with OPA/2-ME, while chondroitin having an acetyl group at the amine group, so we needed deacetylation using natrium hydroxide to break the acetyl group. The mobile phase used tetrahydrofuran 0.25% in water-acetonitrile (87:13) with a flow rate 1.5 mL/min. Analysis conditions have been optimized, validated in terms of accuracy, precision, linearity, selectivity, limit of detection, and limit of quantitation. The results showed average levels of glucosamine hydrochloride and chondroitin sulphate in tablet and cream dosage forms were 92.76%; 96.11% and 101.15%; 100.33% and fulfilled the acceptance criteria

Keywords: glucosamine hydrochloride, chondroitin sulphate, derivatization, OPA/2-ME, HPLC, optimization, fluorescence, validation