The development of new ACL treatment strategies is often initiated with in vitro research. In vitro (Latin for “in glass”) describes techniques where cells are isolated and cultured outside the body to study their response to a variety of specific environmental cues involving scaffolds or growth factors. As such, in vitro studies allow researchers to observe and describe cellular behaviors very precisely under minutely controlled conditions without the necessity of sacrificing research animals. However, the in vitro findings must subsequently be translated into preclinical models to prove that it will also work in vivo before it is translated to clinical research. This chapter briefly describes the basic methodology and provide typical examples of such in vitro models for ACL research.