SUMMARY: 13C-based metabolic flux analysis is a cornerstone of quantitative systems biology, yet its increasing data complexity and methodological diversity place high demands on simulation software. We introduce 13CFLUX(v3), a third-generation simulation platform that combines a high-performance C++ engine with a convenient Python interface. The software delivers substantial performance gains across isotopically stationary and nonstationary analysis workflows, while remaining flexible to accommodate diverse labeling strategies and analytical platforms. Its open-source availability facilitates seamless integration into computational ecosystems and community-driven extension. By supporting multi-experiment integration, multi-tracer studies, and advanced statistical inference such as Bayesian analysis, 13CFLUX provides a robust and extensible framework for modern fluxomics research. AVAILABILITY AND IMPLEMENTATION: Sources and containers are provided at https://jugit.fz-juelich.de/IBG-1/ModSim/Fluxomics/13CFLUX, and scripts to replicate results in the supplementary data at https://github.com/JuBiotech/Supplement-to-Stratmann-et-al.-Bioinformatics-2025.

Science revolves around the best way of conducting an experiment to obtain insightful results. Experiments with maximal information content can be found by computational experimental design (ED) strategies that identify optimal conditions under which to perform the experiment. Several criteria have been proposed to measure the information content, each emphasizing different aspects of the design goal, i.e., reduction of uncertainty. Where experiments are complex or expensive, second sight is at the budget governing the achievable amount of information. In this context, the design objectives cost and information gain are often incommensurable, though dependent. By casting the ED task into a multiple-criteria optimization problem, a set of trade-off designs is derived that approximates the Pareto-frontier which is instrumental for exploring preferable designs. In this work, we present a computational methodology for multiple-criteria ED of information-rich experiments that accounts for virtually any set of design criteria. The methodology is implemented for the case of 13C metabolic flux analysis (MFA), which is arguably the most expensive type among the 'omics' technologies, featuring dozens of design parameters (tracer composition, analytical platform, measurement selection etc.). Supported by an innovative visualization scheme, we demonstrate with two realistic showcases that the use of multiple criteria reveals deep insights into the conflicting interplay between information carriers and cost factors that are not amendable to single-objective ED. For instance, tandem mass spectrometry turns out as best-in-class with respect to information gain, while it delivers this information quality cheaper than the other, routinely applied analytical technologies. Therewith, our Pareto approach to ED offers the investigator great flexibilities in the conception phase of a study to balance costs and benefits.