A novel method for assaying the enzymatic activity of ribosome-inactivating proteins (RIPs) has been developed. The principle of the method is based on that RIP can remove some adenine bases from double-stranded supercoiled DNA molecules, subsequently, the deadenylated DNA was cleaved into nicked and linear form. After treatment with acidic aniline, the deadenylated DNA was degraded into many small fragments, and run out of the gel. The enzymatic activities of two RIPs (trichosanthin and cinnamomin) were tested using this method, the limit of sensitivity is about 50 ng (trichosanthin) and 5 ng (reduced cinnamomin). It should be emphasized that the merit of this method is to avoid the preparation of ribosome.