Stevia rebaudiana leaves consist of non-cariogenic and non-caloric sweeteners (steviol-glycosides) whose consumption could utilize useful effects on human health. The object of this research was to develop and verify liquid chromatography methods with electrospray ionization mass spectrometry (LC-ESI/MS) to evaluate steviol-glycosides or steviol in Stevia leaves. Based on the specific fragmentation of these compounds, an LC–MS/MS method was developed with the aim of quantifying analytes in plant material. The possibility of applying this method was verified in the analysis of stevioside and rebaudioside A from Stevia plants. Finally, on the basis of this metabolomic targeted approach, the results acquired for the samples were handled by Principal Component Analysis, identifying specific genotypic differences based on the geographic origin of the plants.