To obtain meaningful results of hepatic stellate cell (HSC) function, it is crucial to use highly pure HSC populations. Our aim was to optimize HSC isolation from mice livers without exploiting the characteristically transient vitamin A autofluorescence of HSC. HSCs were isolated from C57BL/6 mice using a two-step collagenase digestion and Nycodenz gradient separation followed by CD11b-negative sorting step in order to remove contaminating macrophages and dendritic cells. Isolated cells were analyzed for yield, viability, purity, and potential new markers using immunofluorescence and flow cytometry. We obtained a yield of 350,595 ± 100,773 HSC per mouse liver and a viability of isolated cells of 92.4 ± 3.1%. We observed a low macrophage/dendritic cell contamination of 1.22 ± 0.54%. Using flow cytometry, we demonstrated that CD38 was expressed at the surface of HSC subpopulations and that all expressed intracellular markers specific for HSC in the liver. This isolation method, avoiding fluorescent activated cell sorting (FACS), allowed isolation of HSCs with high purity. Further, flow cytometry analysis suggests that CD38 may be a reliable marker of HSCs and may include subpopulations of HSCs without retinoid droplets.