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Summary: Peritoneal macrophages (PMs) have been shown to have higher stability compared to other macrophage subtypes. However, obtaining enough PMs from a single mouse is often a limitation for metabolomics analysis. Here, we describe a protocol to isolate metabolites from a small number of mouse primary PMs for 13C-stable glucose tracing and metabolomics. Our protocol uses X for metabolite extraction instead of methanol. Our protocol can consistently extract metabolites from low cell number samples with fewer steps than methanol-based approaches.For complete details on the use and execution of this protocol, please refer to De Jesus et al., (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.