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The main objective was to develop a wipe sampling test to measure surface contamination of the most frequently used antineoplastic drugs (ADs) in Swedish healthcare and furthermore to develop an analysis method sensitive enough to assess low levels of contamination. Two wipe sampling tests with separate sample processing methods assessing i) cyclophosphamide (CP), ifosfamide (IF), 5-fluorouracil (5-FU), etoposide (ETO), gemcitabine (GEM) and cytarabine (CYT) (Wipe Test 1), and ii) GEM, CYT and methotrexate (MTX) (Wipe Test 2), respectively were developed by optimization of absorption and extraction efficiencies using different wipe tissue materials, tissue wetting solution and extraction solvents. A fast LC-MS/MS method was developed for simultaneous detection of the studied ADs. The limit of quantification for the method was between 0.04 to 2.4 ng/wipe sample (0.10 to 6.1 pg/cm2 for an area of 400 cm2) and at 50 ng/sample the within-day precision was between 1.3-15%, and the accuracy between 102-127%. Wipe Test 1 was applied in an assessment of cleaning efficiency of five different cleaning solutions (formic acid, water, sodium hydroxide, ethanol and sodium dodecyl sulfate (SDS) for removal of ADs from surfaces made of stainless steel or plastic. For CP, IF, 5-FU, GEM and CYT 92% of the AD were removed regardless of surface and cleaning solution. In conclusion, a user-friendly assessment method to measure low levels of seven ADs in the work environment was developed and validated. Assessment of the decontamination efficiency of cleaning solutions concerning removal of ADs from stainless steel showed that efficiencies differed depending on the AD with water being the least effective cleaning agent. The results suggests that a combination of different cleaning agents including detergent and a solution with an organic component would be optimal to efficiently remove the measured ADs from surfaces in the workplace.