Allosteric ribozymes can be designed to respond to virtually any molecule of choice. The resulting species may be used for example as synthetic regulators of gene expression or alternatively as biosensors. In vitro selection techniques allow the isolation of active molecules from libraries as large as 1015 different molecules. The present protocol describes an in vitro selection strategy for the de novo selection of allosteric self-cleaving ribozymes responding to virtually any drug of choice. We applied this method to select hammerhead ribozymes inhibited specifically by doxycycline or pefloxacin in the sub-micromolar range. The selected ribozymes can be converted into classical aptamers via insertion of a point mutation in the catalytic center of the ribozyme.