Introduction. Glycoalkaloids are plant secondary metabolites that can be both harmful and beneficial to human health. They cause gastroenteric symptoms, coma and even death at high concentrations. It is thought that glycoalkaloids are toxic to human health as a result of their effects on the nervous system and destruction of cell membranes. On the other hand, glycoalkaloids can be effective drugs. For example, solasodine is used to treat skin cancer and tomatidine is used in cancer chemotherapy. Solasodine is also used as a precursor of steroidal drugs. The goal of the work was to isolate and separate efficiently these similar compounds. Materials and methods. Glycoalkaloids from Solanum linnaeanum berries were isolated using column chromatography and confirmed via NMR spectroscopy and MS/MS spectrometry. Results and discussion. The chemical structures of glycoalkaloids are nearly identical. For example, solamargine differs from solasonine in having a methyl group instead of a hydroxyl group bound to the sugar residue of the compound. Thus, their molecular weight and polarity are quite similar. In this work, the process yielded 37.1 mg solasonine, 92.3 mg solasonine and solamargine mix and 56.2 mg solamargine from 81.67 g dried S. linnaeanum berries. Conclusion. Solanum linnaeanum berries are a good source of these glycoalkaloids and the developed protocol proved efficient for purification of solasonine and solamargine.