There is an increasing demand for metabolite analysis for metabolic engineering. However, metabolite analysis is very challenging due to unavailability of sample preparation protocols and lack of highly sensitive analysis methods. This book focuses on metabolite analysis of Clostridium thermocellum which is a thermophilic bacterium that converts biomass to ethanol directly; however, high sensitivity of this bacterium toward ethanol limits its commercial utility. For metabolite analysis, capillary electrophoresis-electrospray ionization-mass spectrometry method (CE-ESI-MS) was utilized due to highly polar and charged nature of metabolites. For metabolite sample preparation, novel methods for quenching and CE compatible metabolite extraction protocols were also developed. The novel sample preparation protocols in combination with CE-ESI-MS technique elucidates the effect of ethanol on metabolite alterations of Clostrodium thermocellum. This book on metabolie analysis on a thermophile will be stepping stone in taking the science of metabolite analysis further and can be used by researchers as reference book.