Human activities induce stress to ecosystems, affecting the key functions provided by microorganisms. Under environmental perturbations and stress, microorganisms reconfigure their metabolism to adapt to the environmental changes. This offers the opportunity to develop chemical tracers that are indicative of microbial stress conditions and their potential to provide ecosystem functions. In this study, we aim to assess changes in ¹⁸O/¹⁶O ratios in cytosolic phosphate (cytosolic δ¹⁸O(PO₄^3-)) as a proxy of microbial metabolic activity level.  δ¹⁸O(PO₄^3-) has been used as an indicator of biological processes; however, which metabolic reactions drive δ¹⁸O(PO₄^3-) remains unknown. To address this challenge, we developed an ¹⁸O stable isotope labeling-based metabolomics workflow to track microbial phosphorylation and identify the key phosphoryl-transfer reactions regulating cytosolic δ¹⁸O(PO₄^3-). Specifically, we cultivated model microorganisms in batch cultures using H₂¹⁸O-labeled media under optimal and heat-stress conditions. By extracting metabolites at different time points and analyzing isotopic incorporation patterns via LC-HRMS, we mapped the differentiated metabolic pathways in the phosphorylation network. The results showed that the redox metabolism associated with oxidative phosphorylation was more responsive to heat stress, while metabolites tied to substrate-level phosphorylation in central carbon metabolism showed only minor or temporal variations. This research supports the use of δ¹⁸O(PO₄^3-) as an indicator of intracellular phosphorylation changes under stress, with broader implications for tracking microbial metabolic responses and phosphorus cycling in changing environmental conditions.