Brassinosteroids (BRs) are a class of naturally occurring phytohormones with poly- hydroxy steroid structure, which regulate general plant growth and many physiological processes. The reported methods for BR analysis were complicated, and the detection sensitivity was relatively low. To realize the automatic analysis of trace BRs in limited plant tissues, an in-tube solid phase microextraction-ultra performance liquid chromatography-tandem mass spectrometry (SPME-UPLC-MS/MS) system was constructed based on two valves-two pumps. Using C18 PEEK column as the trapping column and 4-(dimethylamino) phenylboronic acid (4-DMAPBA) as the derivatization reagent, an on line trapping and in situ derivatization assay method of BRs was developed. BRs could be programmed to fulfill the procedures of injection, extraction, derivatization, LC separation and MS detection in the system. The detection limits of BRs were improved more than one order of magnitude by the online trapping and in situ derivatization techniques, thus endogenous BRs could be quantified in only 300 mg plant tissues.