Gas chromatography-mass spectrometry (GC-MS) is the most frequently applied technique for analysis of Amaryllidaceae alkaloids in plant extracts. These compounds, known for their potent bioactivities, are a distinctive chemotaxonomic feature of the Amaryllidoideae subfamily (Amaryllidaceae). The Amaryllidaceae alkaloids of homolycorine type with a C3-C4 double bond generally show molecular and diagnostic ions at the high mass region with low intensity in EIMS mode leading to problematic identification in complex plant extracts. Eleven standard homolycorine-type alkaloids (isolated and identified by 1D and 2D nuclear magnetic resonance) were subjected to separation with gas chromatography and studied with electron impact mass spectrometry including single quadropole (GC-EIMS), tandem (GC-EIMS/MS), and high resolution (GC-HR-EIMS) detectors, as well as with chemical ionisation mass spectrometry (GC-CIMS). Alkaloid fractions from two Hippeastrum species and Clivia miniata were subjected to GC-EIMS and GC-CIMS for alkaloid identification. GC-EIMS in combination with GC-CIMS provided significant structural information for homolycorine-type alkaloids with C3-C4 double bond facilitating their unambiguous identification. Based on the obtained typical fragmentation, other eleven homolycorine-type compounds were identified in extracts from two Hippeastrum species by parallel GC-EIMS, GC-CIMS and LC-ESI/ToF/MS and in extracts from Clivia miniata by GC-EIMS. GC-MS can be successfully applied for identification of new and known homolycorine-type alkaloids, as well as for chemotaxonomical and chemoecological studies, among others within the Amaryllidoideae subfamily.