In vitro systems are required to evaluate potential liver fibrogenic effects of drugs and compounds during drug development and toxicity screening, respectively. Upon liver injury or toxicity, hepatic stellate cells are activated, thereby acquiring a myofibroblastic phenotype and participating in extracellular matrix deposition and liver fibrosis. The most widely used in vitro models to investigate liver fibrogenesis are primary cultures of hepatic stellate cells, which can be isolated from healthy human livers. Currently, there are no effective methods to maintain hepatic stellate cells in vitro in a quiescent phenotype. Therefore, when cells are plated, they spontaneously become activated in few days. Most in vitro studies in this area have been performed with monocultures of hepatic stellate cells in order to assess the direct effects of a given factor on hepatic stellate cell activation or the induction of inflammatory and fibrogenic responses. In this chapter, focus is put on basic protocols to isolate hepatic stellate cells from human tissue and to maintain them in culture as well as on common in vitro assays to evaluate their response to profibrogenic factors.