To evaluate the anti-inflammatory and anti-oxidant activity of Glyco-Flex® III (GFIII), inflammation was induced using IL-1β in canine chondrocytes. Anti-oxidant capacity was measured using the ABTS method. GFIII whole tablet and separate active constituents (grape seed extract, glucosamine HCI, MSM, DMG, Perna canaliculus) and deracoxib (Deramaxx®) were tested at concentrations 0-1000 μg mL-1. Nitric oxide (NO), soluble collagen (Sircol), sulphated glycosaminoglycan (sGAG), prostaglandin E2 (PGE2), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-3 (MMP-3) were measured using previously established analysis techniques. The anti-oxidant activity of GFIII and constituents was concentration-dependent. GFIII exhibited similar reduction in nitrite, nitrate, soluble collagen, MMP-3 and TNF-α levels than deracoxib at some concentrations, while it has greater potency in reducing PGE2, sGAG and IL-6 at specific concentration ranges. Perna canaliculus has similar potency in reducing nitrite, nitrate, MMP-3 and TNF-α levels than deracoxib, while demonstrating attenuated potency in reducing sGAG, soluble collagen and IL-6 at specific concentration ranges. The other individual constituents exhibited similar pharmacological activity. Glyco-Flex® III whole tablet and components have potent anti-oxidant properties and inhibit cytokine-induced NO and PGE2 production and may ameliorate proteoglycan degradation after an inflammatory insult in vitro.