Establishment of sensitive methods for the detection of cellular sterols and their derivatives is a critical step in developing comprehensive lipidomics technology. We demonstrate that electrospray ionization tandem (triple quadrupole) mass spectrometry (ESI-MS/MS) is an efficient method for monitoring steryl glucosides (SG) and acyl steryl glucosides (ASG). Comparison of analysis of SG and ASG by ESI-MS/MS with analysis by gas chromatography with flame ionization detection (GC-FID) shows that the two methods yield similar molar compositions. These data demonstrate that ESI-MS/MS response per molar amount of sterol conjugate is similar among various molecular species of SG and ASG. Application of ESI-MS/MS to seed samples from wild-type Arabidopsis and a mutant deficient in two UDP-glucose:sterol glucosyltransferases, UGT80A2 and UGT80B1, revealed new details on the composition of sitosteryl, campesteryl and stigmasteryl glucosides and ASG. SG were decreased by 86% in the ugt80A2,B1 double mutant, compared to the wild-type, while ASG were reduced 96%. The results indicate that these glucosyltransferases account for much of the accumulation of the sterol conjugates in wild-type Arabidopsis seeds.