During the past decade, studies on brassinosteroids (BRs) have greatly widened the knowledge of new steroidal plant hormones. This review summarizes studies on BRs from the viewpoints of distribution in plants, bioassays and a microanalytical method using gas chromatography—mass spectrometry with selected ion monitoring (GC-MS-SIM). A highly sensitive and specific bioassay employed to isolate brassinolide from rape pollen is bean-second internode assay. The rice-lamina inclination test and wheat leaf unrolling test are now widely and routinely used mainly in Japan as highly sensitive and specific bioassays during the purification steps of BRs from the plant sources. When a highly purified fraction containing a very small amount of BRs is obtained, the fraction is derivatized with methaneboronic acid to form a bismethaneboronate of BRs and then analysed by GC-MS-SIM. The rice-lamina inclination test and the GC-MS-SIM microanalytical method have contributed greatly to studies on the identification of many natural BRs and also to their screening and distribution in the plant kingdom. So far about 30 natural BRs have been characterized in a number of higher plants (phanerogams) and also in some lower plants (cryptogams). These data strongly suggest that BRs occur widely in the plant kingdom, as in the case of other known plant hormones, and that BRs play some physiological functions in plant growth and development.