Mitochondrial respiration requires redox power that is mainly provided by the tricarboxylic acid (TCA) cycle in heterotrophic tissues. Glycolysis is commonly assumed to be the major pathway for carbon replenishment of the TCA cycle in most plant cells. However, the TCA cycle also provides precursors for amino acids and organic acids synthesis, a process that requires 4- or 5-C molecules supplied by anaplerotic pathways. The TCA cycle is thus involved in both anabolism and catabolism. Despite the good knowledge of enzymes and pathways involved in these processes, the regulation of carbon partitioning between catabolism and anabolism remains poorly understood. Metabolic flux analysis (MFA) aims at quantifying fluxes in metabolic networks and provides new insights for the study of the TCA cycle and associated pathways. This chapter presents briefly the principles of MFA, and describes how C-14-tracing evolved to C-13-MFA, and more recently to C-13-INST-MFA. Such analyses have provided insights about the origin of carbon atoms entering the TCA cycle and the partitioning between respiration and biosyntheses.