The recent legalization of cannabis in many countries and its increased use in scientific and medical research as a result of its therapeutic properties have focused attention on the need for large quantities of highly purified cannabinoids. This is particularly important for the production of (new) analytical-grade standards, used as reference materials, and for the execution of clinical trials to test the biological activity of the single compounds that could potentially be used as new drugs or active pharmaceutical ingredients (APIs). To date, various purification strategies, usually based on several steps, have been proposed with different outcomes. However, all these methods have both advantages and disadvantages. This review will report on the challenges and future perspectives in the purification and isolation of major and minor cannabinoids from cannabis extracts.