Environmental and biological monitoring performed in health care settings during a 14-year period allowed numerous data to be obtained by using the HPLC coupled with tandem mass spectrometry (HPLC-MS/MS). Data was stored in a specific data-base for the assessment of risk exposure to antineoplastic agents (CA). The strategy of analysis was based on the simultaneous determination of different active substances with a lower limit of quantification (LOQ) optimized in the range of sub-units of microg/L. In the present study, the MRM-ESI-MS/MS profiles of seven antineoplastic agents in both environmental and biological matrices are reported. These methods validated according to FDA guidelines allowed our lab to define a profile of antineoplastic agents that was representative of the four IARC classes, such as cyclophosphamide (group 1), cisplatin and doxorubicin (group 2A), daunorubicin (group 2B), 5-fluorouracil, ifosfamide (group 3), taxol, and gemcitabine (group 4). Moreover, contamination levels on a number of work surfaces and trends over a 14-year period are presented. The evaluation of occupational exposure to CA has been based on ALARA principle for most of the past decades but this principle is nowadays overwhelmed by the fast development of technology. The assessment of a possible in-take of CA in hospital personnel, when the sources of environmental contamination are identified, has become possible by the defining of a limit of exposure close to the limit of detection of the analytical method.