Hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass spectrometry (MS/MS) was used to separate artificial and natural sweeteners approved for use in European Union (EU). Among three tested HILIC columns (BlueOrchid PAL-HILIC, Ascentis Express Si and Acclaim™ Trinity™ P2) the last one was selected for the development of HILIC method due to the best results obtained with it. Early eluting and coeluting compounds in HILIC (acesulfame-K, saccharin, cyclamate, sucralose and aspartame) were successfully separated by the HILIC-based approach for the first time. The developed HILIC method allows for determination of all high potency sweeteners in one analytical run. The calibration curves for all analytes had good linearity within the tested ranges. The limits of detection and quantitation were in the range 0.81–3.30 ng/mL and 2.32–9.89 ng/mL, respectively. The obtained recoveries used for trueness and precision estimation were from 98.6% to 106.2% with standard deviation less than 4.1%. Sample preparation was reduced to a necessary minimum and contained only proper dilution and centrifugation. More than twenty samples of beverages were analyzed with the developed HILIC method. Finally, the chromatographic parameters of peaks (reduced retention time, width at baseline, width at 50% of peak height, tailing factor and efficiency) obtained in HILIC mode and in RPLC mode were compared. Developed HILIC method along with RPLC method can be applied for rapid evaluation of sweeteners' content, quality and safety control.