Androgens, produced by Leydig cells in the testis, are essential for male phenotypic differentiation, fertility, and libido. To study the principles and details of Leydig cell function and regulation, in vitro models are crucial. They encompass various experimental approaches such as (i) the testis perfusion technique, (ii) two-dimensional conventional, using primary cultures of purified Leydig cells isolated from the testes of different ages, species, and rodent-origin immortalized tumour Leydig cell lines, and (iii) three-dimensional conditions, such as testicular organotypic or organoid cultures. In an ideal culture condition, Leydig cells maintain their paracrine crosstalk with other testicular cells in a three-dimensional microenvironment, thereby at least partly mimicking the situation in vivo. Today’s understanding of the regulation of Leydig cell steroidogenesis and paracrine/hormonal activity is primarily based on findings obtained from various experimental models in vivo and in vitro, exploiting multiple species, various stages of reproductive development, and different conditions of xenobiotic exposure. The aim of this chapter is to review the main available in vitro techniques and models appropriate for studying molecular and cellular processes associated with steroidogenesis in Leydig cells after exposure to endocrine/paracrine factors and different xenobiotics.