AbstractAn increasing number of genome‐scale reconstructions of intracellular biochemical networks are being generated. Coupled with these stoichiometric models, several systems‐based approaches for probing these reconstructions in silico have been developed. One such approach, called flux balance analysis (FBA), has been effective at predicting systemic phenotypes in the form of fluxes through a reaction network. FBA employs a linear programming (LP) strategy to generate a flux distribution that is optimized toward a particular ‘objective,’ subject to a set of underlying physicochemical and thermodynamic constraints. Although classical FBA assumes steady‐state conditions, several extensions have been proposed in recent years to constrain the allowable flux distributions and enable characterization of dynamic profiles even with minimal kinetic information. Furthermore, FBA coupled with techniques for measuring fluxes in vivo has facilitated integration of computational and experimental approaches, and is allowing pursuit of rational hypothesis‐driven research. Ultimately, as we will describe in this review, studying intracellular reaction fluxes allows us to understand network structure and function and has broad applications ranging from metabolic engineering to drug discovery. Copyright © 2009 John Wiley & Sons, Inc.This article is categorized under: Analytical and Computational Methods > Computational Methods