To explore a simple and easy-to-learn procedure for the isolation of human quiescent hepatic stellate cells (HSCs) that requires no advanced training. Thus reducing costs and increasing efficiency. This protocol will provide sufficient primary cells with minimal contaminants for future basic research on diseases associated with human HSCs. Normal liver tissues were isolated from patients undergoing hepatic hemangioma resection, and a single cell suspension of these tissues was prepared using the Gentle MACS tissue processor. By using this method, the difficulty of the procedure was reduced, fewer cells were lost during the preparation treatments, and the maximal activity of single cells was maintained. Following preparation of the cell suspension, the HSCs were further isolated using a Nycodenz density gradient. Cell viability was examined by trypan blue staining, and the purity of the quiescent human HSCs was determined by autofluorescence and oil red O staining. Activated and quiescent human HSCs were identified using immunofluorescence and Western blotting. The cell cycle distribution in activated and quiescent human HSCs was analyzed by flow cytometry.The recovery rate of the HSCs was approximately (2.1 ± 0.23) × 106 of tissue, with 94.43 ± 1.89% cell viability and 93.8 ± 1.52% purity. The technique used in this study is a simple, high-yield, and repeatable method for HSC isolation that is worthy of recommendation.