Interstitial cystitis and/or bladder pain syndrome (IC/BPS) are characterized by discomfort, abdominal pain, and pelvic pain, and they are often associated with chronic diseases. Pathological conditions related to IC/BPS can occur due to a defect in the integrity of the bladder lining. This defect has been ascribed to damage to the glycosaminoglycan (GAG) layer of the urinary epithelium. In addition, the incipient cascade of inflammation events might prompt extracellular matrix degradation. Several medical devices based on GAG instillation were proposed to re-establish epithelial integrity by GAGs binding to proteoglycans or interacting with structural urothelium. However, to date, only in vitro studies have investigated the GAG, hyaluronic acid (HA). In the present study, TNFα treatment was used to mimic IC/BPS-induced damage in bladder cells in an in vitro model. Highly purified fermentative HA and pharmaceutical grade bovine chondroitin sulfate (CSb), alone or in combination, were evaluated for the ability to counteract bladder cell damage. We evaluated NF-κB with western blots, and we analyzed interleukin 6 and 8 expression at the transcriptional and protein levels with quantitative RT-PCR, western blotting, and ELISA. We also evaluated the expression of an antibacterial peptide, human β-defensin-2. We confirmed our results in a 3D bladder epithelium model. Our results demonstrated that inflammatory status was reduced in the presence of HA, CSb, and the combination of both (HA/CSb 1.6%/2% w/v). This result suggested that these GAGs might be suitable for treating IC/BPS. All the assayed biomarkers showed that HA/CSb treatment modulated cells towards a more physiological status. Finally, we compared two commercial products suggested for the IC/BPS treatments and found that the product with more Ca++, showed enhanced anti-inflammatory activity and provided superior mucoadhesivity.