Metabolomics is defined as a global quantitative assessment of metabolites within a biological system. Metabolic profiling of cell cultures has many potential applications as well as advantages to currently utilized methods for cell-line testing. Metabolite concentrations represent sensitive markers of genomic changes and responses of cells to external stimuli. Effects of drugs or toxins on cell cultures can be observed through the changes in metabolite concentrations. When cell cultures used for production of various biomolecules metabolomics can aid in optimization of cell growth. Metabolomics can also be used as a method for routine monitoring of extracellular metabolic changes in real time measurements. Nuclear magnetic resonance spectroscopy and mass spectrometry are major analytical platforms used for metabolomics measurements. These methods provide detailed, non-biased and highly complementary chemical analyses of metabolic changes within cells (fingerprint) and in excreted metabolites (footprint). This chapter provides review of current applications of metabolomics in cell cultures with an overview of experimental and data analysis methodologies.