The completely unexpected discovery that the RNA molecule has catalytic properties (1 ,2 ) has led to a plethora of interest in the identification and utilization of a variety of catalytic RNA molecules, or ribozymes, that occur in nature. Among others, hammerhead ribozyme is a small catalytic RNA molecule whose catalytic activity resides in a core of less than 40 ribonucleotides (3 ). In its naturally occurring form, the hammerhead ribozyme has the ability to cut itself in a base-specific way. Hammerhead ribozymes can be designed in the laboratory to act in a trans -acting fashion, i.e., to have their catalytic effect on other RNA molecules (4 ). This ability to cleave target RNA molecules can be applied to downregulate unwanted gene expression, a form of genetic therapy. The aim of this chapter is to describe how hammerhead ribozyme activity can be tested in a cell-free environment prior to cell culture and animal experiments. This is an important step in identifying functional ribozymes for use in gene therapy. The following procedures describe the synthesis and testing of hammerhead ribozymes against labeled versions of target RNA molecules.