The alkaloid profile of Hyoscyamus reticulatus L. and Atropa belladonna subsp. caucasica (Kreyer) Avet have not been characterized yet. UHPLC-PDA-Q-TOF-MS/MS and LC-DAD-QqQ-MS/MS methods were used herein to characterize the metabolite profiles of these plants. Flash chromatography in combination with preparative- and semi preparative HPLC were utilized for the isolation of the compounds of interest. The structure of the isolated compounds was proposed based on their MS/MS fragmentation and NMR characteristics. As a total of 19 tropane derivatives, two tyramine derivatives (N-cis- and N-trans- feruloyl tyramine), a lignanamide (grossamide), an alkylamide (pellitorine) were identified in the root and herb extracts of H. reticulatus. Moreover, rutin and caffeoylquinic acids were found in the leaves and fruits, while kaempferol-3-O-glucoside-7-O-rhamnoside and quercetin-3-O-glucoside-rhamnoside-rhamnoside were exclusively characteristic for the leaves of H. reticulatus. The root and herb extracts of A. caucasica contained 16 tropane alkaloid derivatives along with methyl tropate and two tyramine derivatives. © 2022 Informa UK Limited, trading as Taylor & Francis Group.