In this thesis, an analytical platform was designed and applied to various in vitro bacterial and eukaryotic cell cultures. An extraction and an analytical protocol were developed for comprehensive and simultaneous analysis of both lipid and polar metabolites for intra- and extracellular metabolomics using HILIC-LC-TOF-MS. This analytical platform was applied to four diverse research questions such as the effect of oxygen environment on growth, the interplay between gene expression and metabolism, metabolic changes that occur with age, and PAH toxicity. Specifically: (i) the effect of oxygen on the growth, physiology and metabolism of the Gram positive Streptococcus intermedius were investigated by comprehensive intra- and extracellular metabolomes and transcriptome. (ii) Metabolic insights into the role of the multipartite genome of the Gram negative bacteria Sinorhizobium meliloti and its metabolic preferences in a nutritionally complex environment. (iii) Age-associated metabolic dysregulation in murine bone marrow-derived macrophages during bacterial lipopolysaccharide-induced inflammation. (iv) Comprehensive intracellular metabolomic profiles of Sinorhizobium meliloti to sub-lethal exposure of individual or mixtures of polycyclic aromatic hydrocarbon revealed additive and dose-dependent effects. This thesis has demonstrated the versatility of the designed analytical platform and its use for diverse research in cell biology. / Thesis / Doctor of Philosophy (PhD)