Objective:A simple and fast high performance liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) method has been developed for the analysis of brassinosteroids (BRs) in plants without derivatization.Materials:The BRs (including castasterone, 24-epicastasterone, brassinolide and 24-epibrassinolide) have been extracted with ice cold 80% aqueous methanol solution.Method:Five different purification strategies have been tested for the purification and enrichment of BRs.Conclusion:This analytical method was sensitive, reliable, rapid and applicable to trace analysis in complex plant samples.