The plasma concentrations of methotrexate (MTX) and its major metabolite 7-hydroxy methotrexate (7-OH-MTX) are highly correlated with the toxicities in patients with high-dose MTX therapy. Routine monitoring of MTX and 7-OH-MTX plasma levels is useful for dose adjustment of rescue drugs and toxicity prevention. A UHPLC-MS/MS method for simultaneous determination of plasma MTX and 7-OH-MTX was developed, validated, and applied in 181 plasma samples. The ion transition was m/z 455.2 → 308.2 for MTX and m/z 471.2 → 324.1 for 7-OH-MTX. The flow rate was 0.4mL/min with a run time of 2.6min. The calibration range was 0.002–2μM for MTX, and 0.01–10μM for 7-OH-MTX. The intra-day and inter-day inaccuracy and imprecision were –5.50% to 10.93% and less than 9.20% for both analytes. The internal standard (MTX-D3) normalized recovery and matrix factor were consistent at four quality control levels. 14h, 38h, and 62h after dosing, MTX and 7-OH-MTX plasma levels were significantly higher in patients with impaired renal function compared to those with normal renal function. 7-OH-MTX plasma levels were significantly higher in patients with impaired liver function compared to those with normal liver function.