A new method was developed for the quantitative analysis of steryl glycosides in biodiesel (fatty acid methyl esters). This method is much more sensitive than existing methods and has minimum limits of quantification of 50 μg/kg, compared to previously published minimum limits of quantification of about 15 mg/kg. The analysis is based on gas chromatography-mass spectroscopy determination of simple pre-treated and silylated samples via single ion monitoring at 204, 217, 247 m/z, which are specific ions for the silylated sugar moiety. Quantification was carried out using cholesteryl β-D-glucopyranoside as internal standard. The modified synthesis and purification of the internal standard is also presented as well as the characterization by NMR and mass spectroscopy. The advantage of the method compared with other approaches is the simplified sample preparation avoiding extra pre-treatment steps coupled with complete derivatization of the sugar hydroxyl groups by using N,O-bis(trimethylsilyl)acetamide with 5% trimethylchlorosilane as derivatization reagent. On the given conditions high recovery rates ≥ 89% can be obtained. Evaluation of lab specific variance and intermediate precision underline the robustness of the method which will be further assessed by Round robin tests.