AbstractThe search for novel bioactive compounds and the identification of known ones in the plant kingdom are a challenge for the scientists working in different fields of plant science. In the recent years, mass spectrometry is the most frequently applied method for analysis of complex mixtures of plant metabolites. Twenty‐two alkaloids of different lycorine skeleton subtypes (with a Δ3,4 double bond, with a Δ4,11 double bond, with saturated rings C and D, and with aromatic ring C) were subjected to separation with gas chromatography and studied with electron impact mass spectrometry including single quadropole (GC‐EIMS), tandem mass (GC‐EIMS/MS) and high resolution mass (EI‐HRMS) detectors in order to determine their fragmentation pattern. The compounds showed excellent separation and specific MS fragmentation allowing structural determination. The GC–MS can be successfully applied for searching new and identification of known bioactive molecules, chemotaxonomical and chemoecological studies, among others, within the Amaryllidoideae subfamily.