Brassinosteroids (BRs) are a class of steroid plant hormones that participate with other plant hormones in the regulation of numerous developmental processes, including root and shoot growth, vascular differentiation, fertility, and seed germination. A characteristic feature of all plant hormones, including BRs, is that their concentration is extremely low in plant tissues and, therefore, the methods dealing with their determination belong to ultra-trace analysis, for which very sensitive analytical tools are needed. The analysis of natural BRs is essential when their functions and roles in plant growth and development are to be elucidated. Here, we describe a reliable protocol for high-throughput extraction and purification of BRs. The procedure consists of two solid-phase extraction steps and provides selective enrichment and efficient cleanup of these compounds from complex plant extracts. The protocol is designed for sensitive liquid chromatography-tandem mass spectrometry-based method for simultaneous detection of 22 naturally occurring BRs, including their biosynthetic precursors and most of their biologically active metabolites, without need for derivatization.