A new GC-MS method for characterization and quantification of phytosterol oxidation products was developed. Applicability of this method was tested by characterizing sitostanol oxides formed in bulk and then quantifying selected oxides in purified rapeseed oil and tripalmitin matrices in which the complex matrix made oxide analysis difficult. In bulk, nine different sitostanol oxides were characterized, including epimers of 7- and 15-hydroxysitostanol and 6- and 7-ketositostanol. In both lipid matrices, the amounts of sitostanol oxides generated in thermo-oxidation were very low. According to statistical analyses, depending on the oxide, the GC-MS results were the same or slightly higher than those quantified by the more common GC-FID method. Thus, GC-MS provides a powerful alternative for characterization and quantification of phytostanol oxides found in low amounts in complex matrices and is a promising method for future phytosterol oxide studies.