Existing methods for evaluating the potential gonadotoxicity of environmental agents and pharmaceutical compounds rely heavily on animal studies. The current gold standard in vivo functional assays in animals are limited in their human predictive capacity. In addition, existing human 2-dimentional (2D) in vitro models of testicular toxicity do not accurately reflect the in vivo situation. A more reliable testicular in vitro model system is needed to better assess the gonadotoxic potential of drugs prior to progression into clinical trials. The overall goal of this study was to develop a 3-dimensional (3D) in vitro human testis organoid culture system for use as both a predictive first tier drug-screening tool and as a model of human testicular function. Multicellular human testicular organoids composed of spermatogonial stem cells (SSC), Sertoli and Leydig cells were created and evaluated over time for morphology, viability, androgen production and ability to support germ cell differentiation. ELISA measurements confirmed that the organoids produced testosterone continuously with and without HCG stimulation. Upregulating of post-meiotic genes including PRM1 and Acrosin, detected by Q-PCR, digital PCR and Immunofluorescence, indicated the transition of a small percentage of diploid to haploid germ cells. As a novel screening tool for reproductive toxicity, 3D organoids were exposed to 4 antimitotic chemotherapeutic drugs and they responded under all treatment conditions in a dose-dependent manner (different IC50 values). These results indicate that this 3D human testis organoid culture system has the potential to be used as a novel testicular toxicity-screening tool and a novel in vitro model for human spermatogenesis.