Metabolite profiling was conducted to monitor the metabolic differences of the Ralstonia eutropha H16, polyhydroxybutyrate (PHB) producing strain, when grown on different sole carbon sources and to identify the effect of the deletion of the PHB production pathway. The combination of rapid filtration and boiling ethanol extraction successfully extracted intracellular metabolites. Accumulation of acetyl-CoA was detected when the strain was grown on fructose. Whereas, lower adenylate energy charge (AEC) ratio and cell mass was identified on the cells grown on acetate. No significant difference was detected between the wild type and mutant strain except metabolites in the PHB synthetic pathway. Increased PHB production is expected on fructose due to the accumulation of AcCoA, while it is more difficult to improve the production on acetate because of lower metabolic precursor level and AEC ratio.