Objectives: Cyclophosphamide (CP) and ifosfamide (IF) are effective anti-cancer drugs but their genotoxicity can harm everyone contacting them occupationally or environmentally. Therefore, a sensitive method for monitoring their amounts in biological and environmental samples is needed. This has aimed to develop a method for analyzing these drugs in urine and water sewage samples. Material and Methods: The drug spiked samples were extracted, derivatized, and analyzed by gas chromatography–mass spectrometry and the analytical parameters were validated. Results: The method gave linear calibration curves at the concentrations of 0–190 nmol/l. It had the quantitation limit of 3.8 nmol/l and showed acceptable specificity, accuracy, recovery and precision. Conclusions: The developed method can be used reliably for monitoring CP and IF concentrations in urine and water sewage. The method will be applied for preventing health risk from occupational and environmental exposures to these drugs. Int J Occup Med Environ Health 2016;29(5):815–822