Metabolic engineering of industrial microorganisms to produce chemicals, fuels, and drugs has attracted increasing interest as it provides an environment-friendly and renewable route that does not depend on depleting petroleum sources. However, the microbial metabolism is so complex that metabolic engineering efforts often have difficulty in achieving a satisfactory yield, titer, or productivity of the target chemical. To overcome this challenge, 13C Metabolic Flux Analysis (13C-MFA) has been developed to investigate rigorously the cell metabolism and quantify the carbon flux distribution in central metabolic pathways. In the past decade, 13C-MFA has been widely used in academic labs and the biotechnology industry to pinpoint the key issues related to microbial-based chemical production and to guide the development of the appropriate metabolic engineering strategies for improving the biochemical production. In this chapter we introduce the basics of 13C-MFA and illustrate how 13C-MFA has been applied to synergize with metabolic engineering to identify and tackle the rate-limiting steps in biochemical production.