Quantification of brassinosteroids is essential and extremely important to study the molecular mechanisms of their physiological roles in plant growth and development. Herein we present a simple, material and cost-saving high-performance method for determining endogenous BRs in model plants. This new method enables simultaneous enrichment of a wide range of bioactive brassinosteroids (BRs) such as brassinolide (BL), castasterone (CS), teasterone (TE), typhasterol (TY) with ion exchange solid-phase extractions (SPE) and high-sensitivity quantitation of these BRs based on isotope dilution combined with internal standard approach. For routine analysis, the consumption of plant materials was reduced to 1/20 of previously reported and the overall process could be completed within 1 day compared to previous 3 to 4 days. The strategy was validated by profiling BRs in different ecotype and mutants of Rice and Arabidopsis and the BRs distributions in different model plants tissues were determined with the new method. The method allows plant physiologists to monitor the dynamics and distributions of BRs with 1g FW model plant tissues, which will speed up the process for the molecular mechanism research of BRs with these model plants in future work.