Ulcerative colitis is characterized by inflammation, oxidative stress, and excessive free radical production. This study investigated the antioxidant, anti-inflammatory, and protective effects of N-acetylcysteine (NAC), Tiron, and their fixed-ratio combination in acetic acid-induced colitis. An integrated approach was used, combining ligand–ligand docking, acellular antioxidant and protein-denaturation assays, and an in vivo model in male Wistar rats. Colitis was induced by intrarectal administration of 3% acetic acid after 14 days of intraperitoneal pretreatment with NAC, Tiron, or NAC–Tiron. Docking analysis suggested physicochemical compatibility through non-covalent interactions. In vitro, NAC, Tiron, and their combination showed antioxidant and anti-denaturation activities. NAC–Tiron displayed greater activity than the individual compounds in selected endpoints, particularly 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, but did not consistently outperform them across the other assays. In vivo, NAC, Tiron, and NAC–Tiron attenuated macroscopic and histological colonic damage, reduced inflammatory cell infiltration and edema, decreased lipid peroxidation and protein carbonylation, and helped preserve superoxide dismutase (SOD) activity, reduced glutathione (GSH), and total thiols. The treatments also attenuated increases in C-reactive protein (CRP) and free iron without evident worsening of the measured systemic biochemical parameters. Overall, these findings provide an exploratory proof of concept for fixed-ratio NAC–Tiron co-administration but do not establish pharmacological synergy or superiority over standard therapies.