AbstractMetabolic compartmentation represents a major characteristic of eukaryotic cells. The analysis of compartmented metabolic networks is complicated by separation and parallelization of pathways, intracellular transport, and the need for regulatory systems to mediate communication between interdependent compartments. Metabolic flux analysis (MFA) has the potential to reveal compartmented metabolic events, although it is a challenging task requiring demanding experimental techniques and sophisticated modeling. At present no ready‐made solution can be provided to cope with the complexity of compartmented metabolic networks, but new powerful tools are emerging. This review gives an overview of different strategies to approach this issue, focusing on different MFA methods and highlighting the additional information that should be included to improve the outcome of an experiment and associate estimation procedures.