Microwave pretreatment of rapeseeds prior to cold-pressing is a simple and desirable method for producing high quality oils. In this study, a rapid and sensitive lipid profiling platform employing an accurate quantification strategy was established based on direct infusion electrospray ionization tandem mass spectrometry. Using this developed platform, we further investigated the effect of microwave pretreatment of rapeseeds on the contents of important lipids such as triglycerides (TAGs), phospholipids (PLs), and free fatty acids (FFAs) in 15 different rapeseed oils. Our results demonstrated that no significant changes of total FAs and TAGs contents were observed after microwave pretreatment, while FFA contents increased and PLs contents significantly increased up to 40 folds. The potential mechanism of lipid changes was also discussed. The established lipidomics profiling platform can provide reliable lipids profiling data and help to better understand the potential mechanism of microwave pretreatment in oil processing.