Most antineoplastic agents are nonselective in their mechanism of action, affecting noncancerous as well as cancerous cells, and resulting in acute effects such as irritation of mucous membranes and chronic effects such as genotoxicity, teratogenicity, and carcinogenicity. Healthcare workers occupationally exposed to antineoplastic agents are at risk. The present study aimed to develop and apply methods to monitor occupational exposure to antineoplastic agents, using cyclophosphamide (CP) as the model compound. To monitor environmental and biological exposure, potentially contaminated surfaces were wiped and 24 hour urine samples were collected from workers. Liquid chromatography combined with tandem mass spectrometry was performed, with a limit of detection of 0.05 ng/ml. Measurable amounts of CP were detected on 92% of the sampled surfaces, with a geometric mean of 175.22 $ng/m^2$. Despite the environmental contamination of the model compound, CP was below the detection limit in all urine samples. If workplace contamination cannot be completely avoided, it is importance to reduce exposure to the lowest possible levels. To this aim, efforts to minimize occupational exposure along with biological and environmental monitoring are required. The standardized sampling techniques, and specific and sensitive analytical methods reported in this study may be helpful in assessing occupational exposure and devising strategies to reduce exposure.