Abstract Folates are crucial cofactors involved in the de novo generation of purine and deoxythymidine monophosphate, which are essential for DNA synthesis. Antifolates are structural analogues of folate derivatives that act as inhibitors of folate-dependent enzymes and constitute the oldest antimetabolite class of anticancer agents. This review focuses on antifolates with remarkable anticancer activities that include a terminal alkyne function in their molecular structure. The properties of CB3717, a tremendous inhibitor of thymidylate synthase, are described, and the development of raltitrexed and pralatrexate, a dihydrofolate reductase inhibitor approved by the U.S. Food and Drug Administration (FDA) as the first drug for the treatment of relapsed and refractory peripheral T cell lymphoma are presented.