The year 2023 marks the 25th anniversary of the discovery of RNAi. RNAi-based therapeutics enable sequence-specific gene knockdown by eliminating target RNA molecules through complementary base-pairing. A systematic review of published and ongoing clinical trials was performed. Web of Science, PubMed, and Embase were searched from January 1, 1998, to December 30, 2022 for clinical trials using RNAi. Following inclusion, data from the articles were extracted according to a predefined protocol. A total of 90 trials published in 81 articles were included. In addition, ongoing clinical trials were retrieved from ClinicalTrials.gov, resulting in the inclusion of 48 trials. We investigated how maturation of RNAi-based therapeutics and developments in delivery platforms, administration routes, and potential targets shape the current landscape of clinically applied RNAi. Notably, most contemporary clinical trials used either N-acetylgalactosamine delivery and subcutaneous administration or lipid nanoparticle delivery and intravenous administration. In conclusion, RNAi therapeutics have gained great momentum during the past decade, resulting in five approved therapeutics targeting the liver for treatment of severe diseases, and the trajectory depicted by the ongoing trials emphasizes that even more RNAi-based medicines also targeting extra-hepatic tissues are likely to be available in the years to come.