Elementary mode analysis is a useful metabolic pathway analysis tool to characterize cellular metabolism. It can identify all feasible metabolic pathways known as elementary modes that are inherent to a metabolic network. Each elementary mode contains a minimal and unique set of enzymatic reactions that can support cellular functions at steady state. Knowledge of all these pathway options enables systematic characterization of cellular phenotypes, analysis of metabolic network properties (e.g. structure, regulation, robustness, and fragility), phenotypic behavior discovery, and rational strain design for metabolic engineering application. This chapter focuses on the application of elementary mode analysis to reprogram microbial metabolic pathways for rational strain design and the metabolic pathway evolution of designed strains.