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Summary: Mast cells, which constitute tissue-resident immune cells, are distributed in the dural meninges. Here, we provide procedural guidelines for investigating mouse dural mast cells using two techniques. First, we outline the procedures for dural tissue dissection, single-cell isolation, and subsequent surface staining for mast cell identification via flow cytometry. We then describe the techniques employed for whole dura tissue staining to visualize mast cells using confocal and slide scanning microscopy, followed by analysis using Nikon’s NIS-Elements Advanced Research software.For complete details on the use and execution of this protocol, please refer to Lin et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.