〔Abstract〕 Objective To optimize the flow cytometric sorting workflow for myeloid-derived suppressor cells (MDSCs) from murine Lewis lung carcinoma (LLC) subcutaneous tumor tissues, improving single-cell suspension preparation efficiency and sorting purity, thereby providing high-quality cells for subsequent functional studies. Methods Subcutaneous LLC tumor tissues were harvested from C57BL/6 mice, and two enzymatic digestion protocols were first compared to evaluate their effects on single-cell suspension preparation. After staining with a viability dye (Live/Dead) and fluorochrome-conjugated anti-mouse antibodies against CD45, CD11b, and Gr-1, cells were loaded onto a Beckman CytoFLEX cell sorter. Next, conventional and optimized gating strategies were applied. In the optimized strategy, the CD45+ population was used as the initial gate to directly define an MDSC-enriched region, followed by selection of CD11b+Gr-1+ cells. Finally, purity was assessed by flow cytometry, viability was determined by trypan blue staining, and RT-qPCR was performed to measure the expression of MDSC signature genes, including Arg1, Nos2, IL-10, and S100A8/A9. Results The optimized enzymatic digestion protocol significantly increased the yield and viability of single cells. After optimization of the gating strategy, the post-sort positivity rate (purity) was markedly improved. Arg1, Nos2, IL-10and S100A8/A9 were highly expressed in the sorted cells, indicating that the isolated cells retained characteristic MDSC features and functional signatures. Conclusion By optimizing both the tissue digestion protocol and the flow cytometric gating strategy, we establish an efficient workflow for isolating MDSCs from tumor tissues. This method improves the yield and purity of single-cell preparations while preserving antigen integrity, providing a reliable methodological foundation for subsequent studies on MDSC function and metabolism.