Amaryllidaceae alkaloids are a particular group of alkaloids exclusive to the Amarylloideae subfamily. Important from a biological and pharmacological point of view, they have antiparasitic, antiviral and antitumoral activities. Notably, galanthamine has been approved by the FDA as an acetylcholinesterase inhibitory drug for the treatment of Alzheimer's disease. Overall, Amaryllidaceae alkaloids are easy to analyse by GC–MS, but some are difficult to differentiate or detect. In the current study, some of these problems were resolved by applying an alternative analytical technique, high resolution ESI-MS/MS, a soft ionisation method producing different fragmentation patterns. Amongst the Amaryllidaceae alkaloids, only galanthamine has been previously analysed by high resolution ESI-MS/MS. In this work, a large number of Amaryllidaceae alkaloids were studied by high resolution ESI-MS/MS, providing important new structural information.