Sterols are an abundant lipid class in the extraplastidic membranes of plant cells. In addition to free sterols, plants contain different conjugated sterols, i.e. sterol esters, sterol glucosides, and acylated sterol glucosides. Sterol lipids can be measured by gas chromatography after separation via thin-layer chromatography. Here, we describe a comprehensive technique based on the quantification of all four sterol classes by direct infusion quadrupole time-of-flight (Q-TOF) mass spectrometry.