Methotrexate (MTX) is a potent inhibitor of dihydrofolate reductase (DHFR), where is it used as both an antineoplastic and an immunosuppressant. Mechanisms of MTX resistance in cancers include MTX polyglutamylation and upregulation of DHFR. A series of MTX-based PROteolysis TArgeting Chimeras (PROTACs) were designed to selectively degrade human DFHR. These on-target, cell-active PROTACs show proteosome- and E3 ligase-dependent DHFR degradation, selective degradation of DHFR by proteomics, and interpretable structure-activity relationships. Importantly, these PROTACs produced distinct, less-lethal phenotypes compared to MTX, indicating these compounds can complement conventional DFHR enzymatic inhibitors as tool compounds. This chemical probe set, composed of the PROTAC (Diruotrexate), its ester pro-drug (Diruotrexate-ester) and negative control analogs (Diruotrexate-IA1, -IA2), should serve as useful tools for studying one-carbon biochemistry.