Gene function assessment is a main task in biological networking investigations and system biology. High throughput technologies provide an impressive body of data that enables the design of hypotheses linking genes to phenotypes. When a putative scenario is depicted, gene knockdown technologies and RNA-dependent gene silencing are the most frequent approaches to assess the role of key effectors. In this paper, we discuss the relevance of hammerhead ribozymes in target discovery and validation, describing their properties and applications and highlighting their selectivity. In particular, similarities with siRNAs are presented and advantages and drawbacks are discussed. A description of the perspectives of ribozyme application in wide range studies is also provided, strengthening the value of these inhibitors for target validation purposes.