Abstract: OBJECTIVE To establish an UHPLC-MS/MS method for simultaneous quantification of cyclophosphamide(CTX) and its metabolites in rat urine including dechloroethylcyclophosphamide(DCCTX) 4-Ketocyclophosphamide(4-KetoCTX), and carboxyphosphamide(CEPM). METHODS The urine samples were directly diluted by 10% methanol aqueous solution, followed by a centrifugation to get the supernatant for analysis. Chromatographic separation was performed on an Agilent poroshell SB-C₁₈ column(2.1 mm×75 mm, 2.7 μm) using a gradient mobile phase consisting of methanol and 10 mmol·L^-1 ammonium acetate aqueous solution. The buffer flow rate was 0.25 mL·min^-1, and column temperature was maintained at 25 ℃. The protonated ions of analytes were detected in the positive multiple reaction monitoring mode with an electrospray ionization source. The precursor ion and the product ion used for quantitative analysis were: CTX: m/z 261.10→140.10; DCCTX: m/z 199.20→78.00; 4-KetoCTX: m/z 275.10→142.00; CEPM: m/z 293.10→221.10; tinidazole(TNZ): m/z 248.10→121.10. RESULTS Linear ranges for CTX, DCCTX, 4-KetoCTX and CEPM were 40-2 000 ng·mL^-1(r² values were 0.991 5, 0.991 0, 0.995 6, and 0.991 8, respectively). Limit of quantification for all the analytes was 40 ng·mL^-1. The coefficient of RSD for intra-day and inter-day precisions of the quality control samples were 0.67%-12.76% and 1.30%-11.92%. Matrix effect was expressed as internal standard normalized factor. The RSDs all the analytes were between 0.52% and 7.60%. Stability results for all the analytes were in the acceptable levels. CONCLUSION The established method is reliable and reproducible for simultaneous determination of CTX and its metabolites in rat urine after high-dose CTX administration, which is applicable in the cumulative excretion study of these analytes.