The term “metabolome” refers to the entire complement of all the small-molecular-weight metabolites and (generally small-molecule) related species at a given time for a system of interest. Quantitative metabolomic measurements that can cover a significant fraction of either or both intracellular and extra-cellular metabolites are key to understanding metabolism and regulation of cellular systems, and complement gene expression and proteomic information. This coordination of genomic, proteomic, and metabolomic information (also know as “systems biology”) can provide a more complete picture of the permutations occurring within a biological system under varying conditions. Research at PNNL is focusing on the development of ultra-sensitive, quantitative, and high throughput metabolomics measurements based upon the use of high performance liquid chromatography with high accuracy mass spectrometry. We will describe the optimization of sample processing and separations conditions, as well as the application of the accurate mass and time (AMT) tag approach previously developed for high throughput proteomics analyses. Our initial progress will be illustrated by applications to microbial systems.