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Quiescent human hepatic stellate cells (HSCs) serve as important reservoirs of fat-soluble vitamins in the body, namely vitamin A. In an activated form, HSCs are the drivers of fibrosis following chronic liver injury. In non-alcoholic steatohepatitis (NASH) specifically, activated HSCs are drivers of induction and progression of fibrogenesis. Isolation and purification of HSCs from donor liver samples provides an avenue to study HSCs and their fibrotic capabilities. Manual and chemical digestion of donor liver via dissection and Pronase, collagenase, and DNAse treatment creates a suspension of non-parenchymal liver cells. Quiescent HSCs can be further isolated from this suspension by density-gradient centrifugation in a 6%, 8%, 12%, and 15% arabinogalactan medium. After collection of HSCs from the low-density layers of the gradient, they can be grown on uncoated plastic. Rodent HSCs can also be isolated via density-gradient centrifugation. To isolate activated HSCs, liver tissue explants or established immortalized HSC lines can be utilized. Here, we described protocols for isolation of human and rodent HSCs., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)