Metabolic flux analysis (MFA) is a powerful approach for quantifying plant central carbon metabolism based upon a combination of extracellular flux measurements and intracellular isotope labeling measurements. In this chapter, we present the method of isotopically nonstationary ¹³C MFA (INST-MFA), which is applicable to autotrophic systems that are at metabolic steady state but are sampled during the transient period prior to achieving isotopic steady state following the introduction of ¹³CO₂. We describe protocols for performing the necessary isotope labeling experiments, sample collection and quenching, nonaqueous fractionation and extraction of intracellular metabolites, and mass spectrometry (MS) analysis of metabolite labeling. We also outline the steps required to perform computational flux estimation using INST-MFA. By combining several recently developed experimental and computational techniques, INST-MFA provides an important new platform for mapping carbon fluxes that is especially applicable to autotrophic organisms, which are not amenable to steady-state ¹³C MFA experiments.