Folate antagonists are significant molecules and are considered as one of the main categories of antimetabolites. Consequently, they are universally accepted as essential compounds in the pharmaceutical industry. The inhibition of the key enzymes thymidylate synthase (TS) which is a target for antifolate-based chemotherapies and dihydrofolate reductase (DHFR), enables the antifolates to find clinical utility. This perspective highlights and validates the significant role of antifolates in the medicinal chemistry. Recently, considerable efforts have been made to prepare other novel antifolates to improve the antitumor efficacy. In recent reports from our laboratory, we described the preparation of different novel nonclassical antifolates which lack the L-glutamate portion, these compounds exhibited cytotoxicity against various experimental tumors as potently as Methodrexate. The aim of this book was to address the use of the very recently synthetic strategies and their advantages in the synthesis of medically important anticancer antifolates.