Thein vivomeasurement of metabolic flux by¹³C-based metabolic flux analysis (¹³C-MFA) provides valuable information regarding cell physiology. Bioinformatics tools have been developed to estimate metabolic flux distributions from the results of tracer isotopic labeling experiments using a¹³C-labeled carbon source. Metabolic flux is determined by nonlinear fitting of a metabolic model to the isotopic labeling enrichment of intracellular metabolites measured by mass spectrometry. Whereas¹³C-MFA is conventionally performed under isotopically constant conditions, isotopically nonstationary¹³C metabolic flux analysis (INST-¹³C-MFA) has recently been developed for flux analysis of cells with photosynthetic activity and cells at a quasi-steady metabolic state (e.g., primary cells or microorganisms under stationary phase). Here, the development of a novel open source software for INST-¹³C-MFA on the Windows platform is reported. OpenMebius (Open source software for Metabolic flux analysis) provides the function of autogenerating metabolic models for simulating isotopic labeling enrichment from a user-defined configuration worksheet. Analysis using simulated data demonstrated the applicability of OpenMebius for INST-¹³C-MFA. Confidence intervals determined by INST-¹³C-MFA were less than those determined by conventional methods, indicating the potential of INST-¹³C-MFA for precise metabolic flux analysis. OpenMebius is the open source software for the general application of INST-¹³C-MFA.