Objective To establish a UHPLC-MS/MS method for simultaneous determination of cyclophosphamide (CTX) and its metabolites in rat plasma, including carboxyphosphamide (CPM), 4-ketocyclophosphamide (4-Keto CTX), and dechloroethylcyclophosphamide (DC-CTX). Methods Chromatogram separation was performed on an Agilent poroshell SB-C18 (75 mm×2.1 mm,2.7 μm) column using a gradient mobile phase consisting of methanol and 10 mmol/L ammonium acetate aqueous solution. The flow rate was 0.25 mL/min, column temperature was maintained at 25℃, and the injection volume was 5 μL. The protonated ions of analytes were detected in positive ionization under multiple reaction monitoring mode (MRM) with an electrospray ionization (ESI) source. The plasma samples were obtained from eight adult male SD rats to measure plasma concentrations and pharmacokinetic parameters of cyclophosphamide and its metabolites. Results It was showed that the linear relationships of CTX, CPM and 4-Keto CTX were good in the range of 20-4 000 ng/mL (r values were 0.998 0, 0.995 3 and 0.998 6, respectively), and the linear relationship of DC-CTX was good in the range of 5-1 000 ng/mL (r=0.996 8). Relative standard deviation (RSD) of intra-day and inter-day for the quality control (QC) samples and the lower limit of quantitation (LLOQ) samples were lower than 8.73% and 15.38%, respectively. RSD for matrix factor were in the range of -15%-15%, and the recoveries were in the range of (66.44±5.53)%-(96.66±1.73)%. All analytes showed good stability. The rat plasma pharmacokinetic parameters were as follows:C_max of CTX, CPM, 4-Keto CTX and DC-CTX were (207.52±13.20) μg·mL^-1, (18.47±2.66) μg·mL^-1, (6.59±1.33) μg·mL^-1 and (8.27±1.44) μg·mL^-1, respectively; T_1/2 were (1.28±0.09) h, (5.03±0.48) h, (6.72±0.47) h and (7.47±0.68) h, respectively; and AUC_0→t were (372.52±32.79) μg·h·mL^-1, (65.70±5.04) μg·h·mL^-1, (33.26±11.76) μg·h·mL^-1 and (45.03±8.93) μg·h·mL^-1, respectively. Conclusion The established UHPLC-MS/MS method is simple, sensitive, accurate and selective, which makes it suitable for the comprehensive pharmacokinetic study of high-dose CTX in rat plasma.