Four alkaloids, strychnine, soladulcidine, solamargine and (3-O-beta-D-glucopyranosyl-(1 --> 2)beta-D-glucopyranosyl-(1 --> 4)-beta-D-galactopyranoside-(25xi)-solanidan-3beta,23beta-diol)(abbreviation, glycoalkaloid A) were isolated from Solanum lyratum Thunb. The structures were elucidated by NMR and measuring physicochemical properties. Then a novel and rapid method using an ultra-performance liquid chromatography coupled with mass spectrometry was developed and validated for the simultaneous determination of these compounds. An acquit UPLC BEH C18 column (2.1 mm x 50 mm, 1.7 microm) was used. Acetonitrile and 0.1% formic acid were adopted as mobile phase. Detection was performed on a Waters Micromass Quattro Premier tandem quadrupole mass spectrometer in the positive ion mode using an electrospray source. The multiple reaction monitoring (MRM) mode was used to detect the target compounds. The established method showed a good linearity (R2 > 0.999 0) over the investigated concentration ranges, good inter-day and intra-day precisions (less than 2%) and good recoveries (from 99.8% to 100.1%) for all four target compounds. Compared to previous methods employing conventional high performance liquid chromatography (HPLC) separation, the ultra-high-pressure liquid chromatography-tandem mass spectrometry achieved preferable chromatographic parameters and significantly increased sample throughput.