BackgroundThe dermal papilla cells are a specialized population of mesenchymal cells located at the base of the hair follicle (HF), which possess the capacity to regulate HF morphogenesis and regeneration. However, lack of cell-type specific surface markers restricts the isolation of DP cells and application for tissue engineering purposes.MethodsWe describe a novel force-triggered density gradient sedimentation (FDGS) method to efficiently obtain purified follicular DP-spheres cells from neonatal mouse back skin, utilizing only centrifugation and optimized density gradients.ResultsExpression of characteristic DP cell markers, alkaline phosphatase, β-catenin, versican, and neural cell adhesion molecules, were confirmed by immunofluorescence. Further, the patch assays demonstrated that DP cells maintained their hair regenerative capacity in vivo. Compared with current methods, including microdissection and fluorescence-activated cell sorting, the FDGS technique is simpler and more efficient for isolating DP cells from neonatal mouse skin.ConclusionsThe FDGS method will improve the research potential of neonatal mouse pelage-derived DP cells for tissue engineering purposes.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-023-03343-2.