The analysis of plant metabolic networks essentially contributes to the understanding of the efficiency of plant systems in terms of their biotechnological usage. Metabolic fluxes are determined by biochemical parameters such as metabolite concentrations as well as enzyme properties and activities, which in turn are the result of various regulatory events at various levels between control of transcription and posttranslational regulation of enzyme protein activity. Thus, knowledge about metabolic fluxes on a large scale provides an integrated view on the functional state of a metabolically active cell, organ, or system. In this chapter, we introduce flux balance analysis as a constraint-based method for the prediction of optimal metabolic fluxes in a given metabolic network. Furthermore, we provide a step-by-step protocol for metabolic network reconstruction and constraint-based analysis using the COBRA Toolbox. © Springer Science+Business Media New York 2014.