Abstract: The phosphorescent properties of methotrexate and folic acid were studied. Methotrexate phosphoresces Strongly in acidic solution with excitation peak at 346 nm, and emission peak at 454 nm, but it has no phosphorescence in alkaline medium.In comparison with methotrexate, folio acid has intensive phosphorescence both in acid and alkaline media with excitation peaks at 330 and 282 nm and emission peaks at 450 and 422 nm, respectively.The phosphorescent life-time of methotrexate is 0.41 seconds, whereas that of folic acid is 0.22 second. A rapid, simple Phosphorescence method for the determination of methotrexate and folic acid in blood was worked out with the sensitivity of 5 and 10ng respectively.