This study evaluated the effects of adding various doses of taurine and glutathione (GSH) to the rabbit semen extender at 24, 48, and 72 h after cooling at 5°C. Ejaculates with standard color, motility (> 85%), volume, and concentration of 400x106/mL were employed. These ejaculates were diluted with extenders supplemented with 0.5, 1, and 2 mM of GSH and 1, 5, and 10 mM of taurine and chilled at 5 °C. Semen samples were obtained from 20 New Zealand bucks. Non-supplemented samples were used as controls. The levels of total protein, triglycerides, and cholesterol in sperm were measured. The sperm's lipid profile was enhanced by GSH and taurine supplementation by reducing cooling-associated stress, which was determined by maintaining a constant triglyceride concentration. The total protein was maintained by GSH and taurine which was ascertained by keeping triglyceride concentration stable and maintaining the total protein levels in the extender and lowering the cholesterol levels. GSH and taurine supplementation to the extender had protective influences on the in vitro rabbit semen quality during chilled storage for up to 72 h, which were remarkable with increasing supplementation dose and cooling time at 5 °C. The continuity of vitality results from maintaining the vital properties of the cell wall structure by preserving the basic components of the cell wall such as triglyceride, cholesterol, and total protein.