Hyoscyamus boveanus, a rare medicinal herb from the Solanaceae family, is one of seven Hyoscyamus species found in Egypt, and it is the only one endemic to the region. It holds immense medicinal and economic value for the local community. However, it is currently endangered. Thus, the primary goal of this study was to develop a viable in vitro propagation protocol to preserve this valuable species. Surface-sterilized seeds of H. boveanus were germinated on liquid hormone-free Murashige and Skoog (MS) medium. Then, various parts of axenic in vitro grown seedlings (leaves, stems, and roots) were used as explants for both direct and indirect organogenesis. The study examined the effects of different concentrations and combinations of plant growth regulators on the maximum callus induction from these explants in vitro. The hormonal combination of 1.0 mg/L IAA and 0.5 mg/L KIN proved to be the most effective for callus induction, as well as indirect and direct organogenesis among all tested combinations of plant growth regulators. Interestingly, direct organogenesis was observed only in leaf explants from different types of seedlings, whereas all types of seedling explants cultured together in the same media exhibited callus formation, which subsequently developed into roots, shoots, leaves, and flowers. Following this, the plantlets obtained from direct and indirect organogenesis were transferred to asbestos perlite soil growing media. Notably, only the plantlets derived from indirect organogenesis successfully acclimatized, while those from direct organogenesis failed to adapt to the ex-vitro environment.