Abstract: A method for the determination of MTX in biological fluids was described in this paper. Ten rabbits of 2.5～3 kg each was infused with MTX 40～300 mg/kg. Ten patients with malignant tumor aged from 8～53 each received 1～4 g MTX intravenously. A normal hemogram and normal liver and renal functions were prerequisite for this study. After 2～4 hours CF (9～12 mg) was given intramuscularly to rescue every 6 hours for 12 doses.Both MTX in biological fluids of rabbits and patients were extracted and measured with a model-210 UV spcctrophotometer at 306 nm.The equation of the standard curve of MTX was shown to be: Y=0.0456X+0.000092, that of calibrated curve of MTX in plasma was: Y=0.0441X+0.0389, while that of the calibrated curve of MTX in urine was: Y=0.0469X+0.0417. The recovery of MTX by this method was about 95%.For the save use of HD-MTX-CFR, the MTX levels in plasma and it's excretion in urine were also studied.