Brassinosteroids (BRs) are plant steroid hormones that are involved in the regulation of plant growth and development as well as environmental adaptation. The discovery of new BR derivatives is beneficial to their biosynthesis and regulation mechanisms research. However, there are few reports on the methods for exploring new BRs, and the existing methods tend to lack coverage. In this work, we established a comprehensive, highly sensitive and selective structure-oriented method for the screening and structural identification of potential BRs in plants using chemical isotope labeling-assisted liquid chromatography-high resolution mass spectrometry (CL-LC-HRMS). The potential BRs were speculated according to the structural features of the reported BRs, and those speculated BRs containing cis-diol groups were labeled by isotope reagents of 2-methyl-4-phenylaminomethylphenylboronic acid (2-methyl-4-PAMBA) and 2-methyl-4-PAMBA-d5 to improve the sensitivity and selectivity of MS detection. In addition, the fragmentation of 2-methyl-4-PAMBA-labeled BRs via collision-induced dissociation (CID) led to the generation of reporter ions, which contributed to specific screening of potential BRs. In-depth structure of potential BRs was elucidated by analyzing multistage MS (MSn) fragmentation patterns. Using our proposed method, a total of 16 potential BRs were detected from plant samples including 5 new ones, of which one new BR derivative was identified as 2-deoxy-3-epi-6-deoxy-dolichosterone, and this new BR may be involved in the biosynthesis of BRs as precursor of 6-deoxo-dolichosterone. The method developed in this work is promising for screening and identifying new BR derivatives in plants, thereby supplementing the biosynthesis pathway of BRs.