Environmental stressors such as marine heatwaves increasingly threaten coral reef ecosystems worldwide. Coral resilience is largely determined by their algal symbionts (Symbiodiniaceae), making it crucial to understand the metabolic adaptations of coral symbionts. While metabolomics offers powerful insights into cellular mechanisms, traditional approaches have provided limited coverage of symbiont metabolism, particularly for central carbon pathways. We developed a targeted metabolomics workflow optimized for coral symbionts, focusing on polar metabolite analysis through complementary chromatographic techniques. Our approach utilises anion-exchange chromatography's unique capability to resolve highly charged metabolites from symbiont central carbon metabolism, complemented by orthogonal separation methods. This analytical strategy enables detailed investigation of symbiont metabolic networks using minimal biomass. By incorporating dual-labeling experiments with both stable isotope tracers (13C) and deuterium oxide (D2O), we can simultaneously monitor metabolite flux and biosynthetic rates within symbiont cells. This methodology reveals previously undetectable metabolic patterns in symbiont response to environmental stress, providing new insights into their metabolic plasticity. Understanding these fundamental aspects of symbiont metabolism advances our knowledge on these crucial organisms’ respond to environmental change and provides targets for bio-engineering solutions focusing on coral symbionts.