Organoid systems take advantage of the self-organizing capabilities of cells to create diverse multi-cellular tissue surrogates that constitute a powerful novel class of biological models. Clearly, the formation of a testicular organoid (TO) in which human spermatogenesis can proceed from a single-cell suspension would exert a tremendous impact on research and development, clinical treatment of infertility, and screening of potential drugs and toxic agents. Recently, we showed that primary adult and pubertal human testicular cells auto-assembled in TOs either with or without the support of a natural testis scaffold. These mini-tissues harboured both the spermatogonial stem cells and their important niche cells, which retained certain specific functions during long-term culture. As such, human TOs might advance the development of a system allowing human in vitro spermatogenesis. Here we describe the methodology to make scaffold-based and scaffold-free TOs.