Introduction: Veratrum alkaloids have gained attention due to their toxic effects and potential pharmaceutical applications, particularly in cancer and cardiology. Over 200 alkaloids are found in species of the Veratrum genus. The alkaloid composition and concentrations can greatly vary in plants depending on factors like species, plant part, location, season, weather, or nutrients. Objective: This study aims an analytical approach to analyze and quantify Veratrum alkaloids in different plant parts of Veratrum species. The purpose is to contribute essential alkaloid concentration data for future research on the pharmacological and toxicological aspects of Veratrum alkaloids. Methods: This study focuses on five Veratrum alkaloids (cevadine, jervine, protoveratrine A, veratramine, and veratridine) in three Veratrum species (Veratrum album L., Veratrum californicum Durand, and Veratrum nigrum L.) collected from four German botanical gardens (Dresden, Leipzig, Marburg, and Schellerhau). A liquid–liquid extraction method and a sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) method operating in multiple reaction monitoring (MRM) mode were applied for the alkaloid determination. Results: Quantification revealed varying alkaloid concentrations among plant parts and Veratrum species in the μg/g to mg/g range. Protoveratrine A exhibited the highest content, while veratramine concentrations were generally lower. Especially in fruit, roots and rootstock of Veratrum album L. alkaloid concentrations were significant high. Conclusion: The developed HPLC-MS/MS method successfully determined Veratrum alkaloid concentrations in plant samples. The study contributes valuable data on Veratrum alkaloid distribution in different species and plant parts, crucial for understanding their potential medicinal and toxicological significance.