Post-translational modification of proteins by reversible phosphorylation plays a pivotal role in regulating vital cellular processes. Despite the importance of the phosphorylation level of regulation, little work has been carried out on the phosphoproteomic characterization of Chinese hamster ovary (CHO) cells in bioprocess-relevant conditions. To bridge this knowledge gap, through application of mass spectrometry-based proteomics and phosphoproteomics, I present three original, studies that investigate (1) the dynamic nature of the CHO cell phosphoproteome in response to changing culture conditions; (2) differential activation of transcription factors between cell lines of varying specific productivity; and (3) changes in the CHO cell proteome and phosphoproteome in response to adaptation to growth in glutamine free media. Study 1 shows that the inclusion of phosphoproteomic data significantly improves proteome coverage and gives insights into cell signalling pathways that could be targeted to control growth. Study 2 shows that the nuclear proteome and phosphoproteome have an essential role in regulating the final productivity of recombinant proteins from CHO cells and that CREB1 may play a role in transcriptional enhancement. Finally, study 3 provides a comprehensive proteomic and phosphoproteomic analysis of how cellular proteome expression changes after adaptation to glutamine-free growth conditions and highlights critical pathways to consider when designing future studies to further understand and engineer glutamine metabolism, and rational design of improved feeding strategies. Together, these studies advance our understanding of CHO cell biology and provide new avenues for exploration of targets for cell line engineering to improve the efficiency of production of recombinant biotherapeutics.