INTRODUCTION: Cyclotides are plant-made defence proteins with a head-to-tail cyclic backbone combined with a conserved, six cystine knot. They have a range of biological activities, including uterotonic and anti-HIV activity, which have attracted attention to their potential pharmaceutical applications. Furthermore, their unique structures and high stability make them appealing as peptide-based templates for drug design applications. Methods have been developed for their production, including solid phase peptide synthesis as well as recombinant methods. AREAS COVERED: This article reviews the recent literature associated with therapeutic applications of naturally occurring and synthetically modified cyclotides. It includes applications of cyclotides and cyclotide-like molecules as peptide-based drug leads and diagnostic agents. EXPERT OPINION: The ultra-stable cyclotides are promising templates for drug development applications and are currently being assessed for the potential breadth of their applications. For synthetic versions of cyclotides to enter human clinical trials further studies to examine their biopharmaceutical properties and toxicities are required. However, several promising proof-of-concept studies have established that pharmaceutically relevant bioactive peptide sequences can be grafted into cyclotide frameworks and thereby stabilised, while maintaining biological activity. These studies include examples directed at cancer, cardiovascular disease and infectious diseases. Solid phase peptide synthesis has been the preferred approach for making pharmaceutically modified cyclotides so far, but promising progress is being made in biological approaches to cyclotide production.