BACKGROUND: The alkylating agent cyclophosphamide is used in chemotherapy regimens for various type of cancer. However, cyclophosphamide may lead to toxic side effects on the bladder, namely hemorrhagic cystitis, which can cause hematuria, and, potentially, bladder cancer. These effects are caused by acrolein, a byproduct of cyclophosphamide metabolism. In this study, a method to quantify 3-hydroxypropyl mercapturic acid (3-HPMA) in urine was developed. 3-HPMA is a stable metabolite of acrolein that serves as biomarker of acrolein. METHODS: Urine samples were collected 4 hours after cyclophosphamide administration and analyzed to determine the risk of hematuria. 3-HPMA was analyzed by reverse-phase LC-MS/MS using a triple quadrupole electrospray ionization mass spectrometer in the positive-ion mode. The mobile phase was a 90:10 (vol/vol) mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Multiple reaction monitoring mode was used, with m/z 222.10 → 90.97 for 3-HPMA and 164.10 → 122.02 for the internal standard N-acetyl cysteine (NAC). Samples were prepared by acidification and dilution. RESULTS: The analytical method produced a linear response within the concentration range of 40-10,000 ng/mL. The method was validated in accordance with 2018 FDA guidelines and applied to quantify 3-HPMA in the urine of 40 patients with breast cancer. The measured concentrations ranged from 820.3 to 5596.1 ng/mg creatinine. Seven patients identified with hematuria had low 3-HPMA concentrations of 4445.824 ± 411.17 ng/mg creatinine, and 33 patients without hematuria had low 3-HPMA concentrations of 2419.4 ± 1171.8 ng/mg creatinine. CONCLUSIONS: The method was applicable for the quantification of 3-HPMA in human urine. Large variations in 3-HPMA concentrations were found in 40 patients with breast cancer treated with cyclophosphamide, with a significant difference (P < 0.05) observed between patients with hematuria and those without hematuria.