The analysis of metabolic differences in bacterial strains is a useful tool for the development of strains with desired growth and production properties. Several methods are available for the evaluation and understanding of the differences: Biochemical methods to measure metabolites concentration and enzyme activity, mathematical methods to analyze metabolic fluxes through the various pathways, proteomic methods to identify expressed proteins, and genomic methods to detect and measure gene expression. A combination of the various methods is required to obtain a comprehensive understanding of metabolic activities. The genomic methods provide substantial amount information on global gene expression but do not always reflect the actual activity of the individual components. The review focuses on the different methodologies and their use, as well as historical overview of the evaluation of the differences between Escherichia coli K and E. coli B.