The Solanaceae plant family contains at least 98 genera and over 2700 species. The Duboisia genus stands out for its ability to produce pyridine and tropane alkaloids, which are relatively poorly characterized at the phytochemical level. In this study, we analyzed dried leaves of Duboisia spp. using supercritical CO2 extraction and ultra-high-pressure liquid chromatography coupled to high-resolution tandem mass spectrometry, followed by feature-based molecular networking. Thirty-one known tropane alkaloids were putatively annotated, and the identity of six (atropine, scopolamine, anisodamine, aposcopolamine, apoatropine, and noratropine) were identified using reference standards. Two new granatane alkaloids connected in the molecular network were highlighted from Duboisia myoporoides, and their α-granatane tropate and α-granatane isovalerate structures were unambiguously established by semisynthesis.