This HPTLC Application Tutorial deals with the quantitative determination of steviol glycosides in sweeteners. The steps shown in the tutorial include application with ATS 4, development with ADC 2, derivatization by dipping and heating, documentation with the TLC Visualizer, scanning with TLC Scanner 4 and identification by mass spectrometry with the TLC-MS Interface coupled to a single-quadrupole mass spectrometer. All instrumental steps, except identification, are controlled by visionCATS.