Background: Solanum trilobatum L. (Solanaceae) is an endangered medicinal plant valued for its steroidal alkaloids, solasodine (anticancer) and diosgenin (steroid precursor). Conventional cultivation cannot meet pharmaceutical demands due to ecological threats.

Methods: An optimized in vitro protocol was developed using leaf explants on MS medium with 0.1 mg/L IAA + 2.5 mg/L kinetin. Solasodine and diosgenin were isolated from in vitro and field-grown plants, characterized via TLC, UV-Vis, and IR spectroscopy, and tested for cytotoxicity against DLA cells.

Results: Maximum shoot proliferation (32 shoots/explant) was achieved. In vitro-derived solasodine showed 78–82% cytotoxicity (100 µg/mL), matching field-grown plants, while yielding 28% higher content (3.2 vs. 2.5 mg/g DW).