Background: Flucloxacillin is a widely used antibiotic but data are lacking on the risks associated with occupational exposure. Aim: To evaluate flucloxacillin stability; to identify an effective decontamination reagent for flucloxacillin; and to identify glove material that will prevent flucloxacillin permeation. Method: Decontamination reagents used for flucloxacillin wipe sampling and degradation/stability tests included: isopropanol 50% and 70%, ethanol 60% and 100%, benzalkonium chloride 0.1% (Pine O Cleen), sodium hypochlorite 0.5% (Chloroclens), sodium hydroxide 0.03M and distilled water. Short-term (0 to 90 minutes) and daily degradation (0 to 6 days) of flucloxacillin in these media and flucloxacillin recovery from surface wipe sampling was investigated using high pressure liquid chromatography with ultraviolet detection. Flucloxacillin permeation was tested using disposable, powder-free latex and nitrile gloves in a disposable test cell to which solution was applied on one side of the glove material and wiped from the other side after 3 hours. Results: This analytical method for assessing flucloxacillin contamination has a limit of detection of 0.1 µg/mL. Distilled water was the most suitable solvent as degradation of flucloxacillin was minimal. Sodium hypochlorite 0.5%, followed by sodium hydroxide 0.03M and benzalkonium chloride 0.1%, were the most effective decontamination reagents. No permeation of flucloxacillin was detected through the latex or nitrile gloves after a 3 hour period. Conclusion: A method for assessing occupational exposure to flucloxacillin is described in this paper. Sodium hypochlorite 0.5% was the most effective decontamination reagent for flucloxacillin. Both the latex and nitrile gloves prevented flucloxacillin permeation for up to 3 hours. J Pharm Pract Res 2009; 39: 95-8.