Natural compounds occur as various isomeric or closely related structures in biological matrices. These compounds are difficult to separate from the complex mixtures, and hence, the need for effective and innovative separation techniques arises. Recycle HPLC allows the recycling of sample, in part or full, and increases the separation efficiency of the process while keeping the peak dispersion to a minimum. Recycling in an HPLC system has been used in the isolation and purification of different types of natural products including enantiomers, diastereomers, epimers, positional isomers, and structurally related or unrelated compounds having similar retention characteristics. The present paper overviews the development of instrumentation and setup of recycle HPLC and its applications in the separation of natural products.