Chinese hamster ovary cells (CHO) were exposed to highly concentrated feed solution during a fed-batch cultivation which causes an unphysiological osmolality increase (>300 mOsm/kg) affecting cell physiology, morphology and proteome. To get a deeper insight into underlying molecular mechanisms involved in cellular hyperosmolality response, we performed a comparative quantitative label-free proteome study of hyperosmolality-exposed vs. control CHO cells by nanoLC-ESI-MS measurement approach (orbitrap-MS). Our observations correlate well with the data from recent CHO-based fluxome and transcriptome studies and expose hitherto unknown targets involved in response to hyperosmotic pressure in mammalian cells.