For over three decades, Chinese hamster ovary (CHO) cells have been the chosen expression platform for the production of therapeutic proteins with complex post‐translational modifications. However, the metabolism of these cells is far from perfect and optimized, and requires substantial know how and process optimization and monitoring to perform efficiently. One of the main reasons for this is the production and accumulation of toxic and growth‐inhibiting metabolites during culture. Lactate and ammonium are the most known, but many more have been identified. In this review, an overview of metabolites that deplete and accumulate throughout the course of cultivations with toxic and growth inhibitory effects to the cells is presented. Further, an overview of the CHO metabolism with emphasis to metabolic pathways of amino acids, glutathione (GSH), and related compounds which have growth‐inhibiting and/or toxic effect on the cells is provided. Additionally, relevant publications which describe the applications of metabolomics as a powerful tool for revealing which reactions occur in the cell under certain conditions are surveyed and growth‐inhibiting and toxic metabolites are identified. Also, a number of resources that describe the cellular mechanisms of CHO and are available on‐line are presented. Finally, the application of this knowledge for bioprocess and medium development and cell line engineering is discussed. Chinese hamster ovary (CHO) cells are the chosen expression platform for the production of therapeutic proteins. ‘Omics data sets can aid and guide the rational design and generation of mammalian cells factories free of unwanted metabolic products and therefore capable of achieving higher cell densities and productivities. In this review, a number of metabolites depleting and accumulating throughout the course of cultivations, that have toxic and growth inhibitory effects to the cells are presented. Additionally, the potential application of this knowledge along with the use of analytical methods and on‐line metabolic resources for enhancing CHO cell lines by combining media development and cell line engineering approaches is discussed.