Chinese Hamster Ovary (CHO) cells are widely utilised in the biopharmaceutical industry to produce therapeutic proteins. Understanding the mechanisms of endoplasmic reticulum (ER) stress and its interplay with protein degradation pathways remains pivotal for improving production efficiency and product quality. Proteasome inhibitors such as MG132 offer a valuable tool for studying ER stress by disrupting protein degradation and promoting the accumulation of ubiquitinated proteins. In this study, we investigated the proteomic responses of CHO-K1 (non-producer), CHO DP-12 and NISTCHO (IgG producer) cell lines under ER stress induced by a combination of the proteasome inhibitor MG132 and the glycosylation inhibitor tunicamycin. The combined treatment of MG132 and tunicamycin intensified ER stress by preventing the degradation of ubiquitinated misfolded proteins, activating the unfolded protein response (UPR) more robustly than either agent alone. Key pathways related to protein folding, ER- associated degradation (ERAD), and the ubiquitin-proteasome system were significantly altered, particularly in the producer cell line NISTCHO. By modulating stress responses and protein degradation pathways, this work contributes to improving the yield and quality of biopharmaceuticals in industrial production settings.