Methotrexate has been a clinical agent used in cancer, immunosuppression, rheumatoid arthritis, and other highly proliferative diseases for many years, yet its underlying molecular mechanism of action in these therapeutic areas is still unclear. We have previously reported using a chemical proteomics technique on several other potential pharmacodynamic targets of methotrexate. Here, using a frontal affinity chromatography with mass spectrometry detection, we confirm one of these targets, hypoxanthine-guanine amidophosphoribosyltransferase, as a true binder of methotrexate with a K d of 4.2 M. These results complement and confirm our recent study, but more importantly, shed light into the mechanism of action of methotrexate in oncology and other highly proliferative diseases and may help explain some unaccounted for effects of this drug. For example, despite the fact that DNA salvage pathway enzymes are highly active, methotrexate can be effective if it only targets enzymes of the de novo pathway.