Industrial bioprocesses place extraordinary demands on the intermediary metabolism of host cells to meet the biosynthetic requirements for maximal growth and product formation. Therefore, engineering cell metabolism to promote high yield and specific productivity is a major goal of the biotech industry. 13C metabolic flux analysis (MFA) provides a rigorous approach to quantify host metabolic phenotypes by applying isotope tracers to map the flow of carbon through intracellular biochemical pathways. We have previously developed a package of MATLAB routines called INCA that automates the computational workflow of MFA. I will discuss two ongoing studies where my lab has applied INCA to identify targets for metabolic engineering of host cell factories. First, we performed 13C MFA to characterize the metabolism of IgG-expressing CHO cells during four separate phases of a fed-batch culture. During stationary phase when IgG production peaked, energy was primarily generated through CAC and oxidative phosphorylation. We next examined nine CHO clones cultured in 3-liter fed-batch bioreactors, to further assess their metabolism during stationary phase. The lactate dehydrogenase (LDH) flux was most closely associated with specific IgG productivity: as IgG productivity increased, lactate production decreased. Additionally, elevated CAC fluxes corresponded strongly with increased IgG specific productivity. We are currently developing strategies to engineer these high-productivity metabolic phenotypes to develop next-generation CHO hosts for protein biomanufacturing. Second, we applied 13C MFA to analyze the photosynthetic metabolism of cyanobacteria strains that have been engineered to produce the industrial biochemical isobutyraldehyde (IBA). The flux analysis identified a potential bottleneck at the pyruvate kinase (PK) reaction. Based on these results, we overexpressed PK to engineer strains with significant improvements in IBA production. 13C flux analysis of the resulting strains identified further targets for debottlenecking IBA production in cyanobacteria, some of which are currently under investigation. These studies have established 13C MFA and the INCA software package as a comprehensive platform to map carbon fluxes in animal and microbial cell cultures used for biomanufacturing.