Abstract: In this paper, DEAE ion-exchange column chromatography was used for separation of MTX and its major metabolite, 7-OH-MTX, from biological fluids of rabbits and tumor patients with a high-dose MTX of 100mg/kg for rabbit, and 2.0g/tumor patient. Their absorptivity values was determined by ultraviolet absorption at 302 nm and 305nm, respectively. Maximum sensitivity achieved was 1×10^-7 M. The recovery of MTX and 7-OH-MTX was 93.6%.The method overcome shortcoming of other methods such as: total-radioactivityassay, fluorescence assay, radioimmunoassay, enzyme inhibition and others, all of which cannot distinguish MTX from 7-OH-MTX, and folic acid analogs.Our work offered experimental basis for-clinical-pharmacological investigations.