Increasing demand of protein biotherapeutics produced using Chinese hamster ovary (CHO) cell lines necessitates improvement in the production yield of the bioprocess. Various cell engineering, improved media formulation and process-design based approaches utilizing the power of OMICS technologies, specifically, genomics and proteomics, have been employed; however, the potential of metabolomics largely remains unexplored. Metabolomics enables the detection, identification, and/or quantitation of small molecules, commonly known as metabolites, in and around the cells and may help to unlock the cellular molecular mechanism(s) that regulates cell growth and productivity in the bioprocess and improves cellular performance during the bioprocess. Currently, liquid chromatography (LC)/gas chromatography (CG)- coupled with mass-spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are the most commonly used approaches for metabolomics. Therefore, in this chapter, we have discussed the standard procedures of investigating CHO metabolites using LC/GC-MS and/or NMR-based approaches.