Cytotoxic antineoplastic drugs (ADs) pose occupational risk and therefore require safe handling practices. We created, optimised, and validated an innovative monitoring protocol for simultaneously assessing 21 ADs in the healthcare environment, and also proposed surface exposure levels (SELs) to facilitate the interpretation of monitoring results, as there are currently no occupational exposure limits for ADs. The environmental AD monitoring data were collected in nine Italian hospitals between 2008 and 2017 and include 74,565 measurements in 4,814 wipe samples. Excellent overall recovery and sensitivity of the analytical methods along with innovative desorption automation make this protocol useful for routine monitoring. AD contamination was found in 3,081 measurements, confirming potential exposure in healthcare workers. Samples taken at the beginning and the end of work shifts, allowed to calculate 75th and 90th percentile values for each ADs both in preparation and administration units and we created a traffic-light colour-coding system to facilitate interpretation of the findings. The introduction of SELs will provide a solid basis for improving occupational safety and focusing on contamination control.