Galanthus x valentinei (J. Allen) Beck nothosubsp. subplicatus (N. Zeybek) A. P. Davis (Amaryllidaceae) is an endemic hybrid between G. nivalis L. and G. plicatus M. Bieb. subsp. byzantinus (Baker) D. A. Webb. Lycorine, a common alkaloid found in Amaryllidaceae plants, has been shown to display important biological activities. In the present study, a reversed-phase high-performance liquid chromatographic method has been used for the quantitative determination of lycorine in the aerial parts and bulbs of G. xvalentinei nothosubsp. subplicatus. A simple method for the extraction of lycorine in lowmass plant samples was employed utilizing pre-packed columns with diatomaceous earth (Extrelut®). The chromatographic separation was carried out using an isocratic system with a mobile phase of trifluoroacetic acid-water-acetonitrile (0.01:95:5) applied at a flow rate of 1 mL min −1 using diode array dedector. The content of lycorine in the bulbs of G. xvalentinei nothosubsp. subplicatus was found to be 0.0028%, however, it was not detected in the aerial parts of this hybrid.