Metabolic Flux Analysis (MFA) uses a stoichiometric model of intracellular reactions and measured extracellular rates (substrate uptake, product secretion, O₂ consumption, CO₂ evolution) to calculate intracellular fluxes by solving the mass balance equation: $\mathbf{S} \cdot \mathbf{v} = 0$ at pseudo-steady state. ¹³C-MFA uses isotope labelling to resolve fluxes in parallel pathways. MFA is essential for identifying bottlenecks in metabolic engineering projects.