Biotoxin activity assays typically involve multistep sample preparation, multicomponent reactions, multistep analysis, or a combination thereof. We report a single-step, real-time ricin activity assay that requires little or no sample preparation and employs direct analysis in real time mass spectrometry. The release of adenine from the inhomogeneous substrate herring sperm DNA by ricin was determined to be 53 +/- 2 pmol adenine per picomole of ricin per hour. This procedure can be readily adapted to any enzyme for which a reactant or product of low molecular weight (up to approximately 600) can be identified.