BACKGROUND: Germ cell development involves formation of the spermatogenic or oogenic lineages from the bipotential primordial germ cells. Signaling mechanisms in the fetal testis and ovary determine whether germ cells enter the male or female developmental pathway, respectively. These signaling processes underpin an important phase of germ cell development, disruption of which can lead to failed germ cell function resulting in infertility or the formation of germ cell tumours. FINDINGS: In this study we have developed a small molecule screening protocol combined with flow cytometry to identify signaling pathways that direct male-specific development of germ cells. Here we provide a detailed method for this screening protocol, which we have used to identify signaling pathways important for male germ cell development. CONCLUSION: This method will be of particular use in screening inhibitors of signaling pathways, endocrine disruptors or other chemicals for their ability to disrupt testis and germ cell development, thereby providing insight into testicular dysgenesis and factors underlying poor male reproductive health.