A sensitive, specific and accurate high-performance liquid chromatography-ion spray-tandem mass spectrometry procedure (HPLC/MS/MS) has been developed to quantify cyclophosphamide in human urine. This methodology, which includes the liquid-liquid extraction with ethyl acetate, requires no derivatization procedures, preventing cyclophosphamide from possible thermal and chemical decomposition reactions. This methodology was validated by the use of ifosfamide as internal standard (I.S.). The assay was linear over the range 0 to 3.2 ng mL-1 urine, having a low limit of quantification of 0.2 ng mL-1. The low limit of detection was assessed at 0.05 ng mL-1 urine. This method is characterized by a coefficient of variation < 10%. Standard calibration curves, performed on three different days, had correlation coefficient always greater than 0.998. The intra and inter-day precision were within 11%, and accuracy was included in the range 99-103%. The mean extracted recovery assessed at three different concentrations (0.5, 0.8, 3.2 ng mL-1) was always more than 85%. The extraction efficiency of cyclophosphamide from urine samples was also studied at six different pH values (pH 4, 5, 6, 7, 8, 10). CP gave the maximum extraction efficiency when pH urine solutions was adjusted to 7.0 and somewhat lower at the other considered values.