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13 C-metabolic flux analysis ( 13 C-MFA) allows metabolic fluxes to be quantified in living organisms and is a major tool in biotechnology and systems biology. Current  13 C-MFA approaches model label propagation starting from the extracellular  13 C-labeled nutrient(s), which limits their applicability to the analysis of pathways close to this metabolic entry point. Here, we propose a new approach to quantify fluxes through any metabolic subnetwork of interest by modeling label propagation directly from the metabolic precursor(s) of this subnetwork. The flux calculations are thus purely based on information from within the subnetwork of interest, and no additional knowledge about the surrounding network (such as atom transitions in upstream reactions or the labeling of the extracellular nutrient) is required. This approach, termed ScalaFlux for SCALAble metabolic FLUX analysis, can be scaled up from individual reactions to pathways to sets of pathways. ScalaFlux has several benefits compared with current  13 C-MFA approaches: greater network coverage, lower data requirements, independence from cell physiology, robustness to gaps in data and network information, better computational efficiency, applicability to rich media, and enhanced flux identifiability. We validated ScalaFlux using a theoretical network and simulated data. We also used the approach to quantify fluxes through the prenyl pyrophosphate pathway of  Saccharomyces cerevisiae  mutants engineered to produce phytoene, using a dataset for which fluxes could not be calculated using existing approaches. A broad range of metabolic systems can be targeted with minimal cost and effort, making ScalaFlux a valuable tool for the analysis of metabolic fluxes.

ScalaFlux: A scalable approach to quantify fluxes in metabolic subnetworks