Allergic reactions to beta-lactams, such as penicillin, can be life-threatening. Due to the large number of individuals allergic to beta-lactams, a method for their inactivation was explored such that a contaminated area could be treated and re-used. The goal was to validate a cycle that could be used to treat a pharmaceutical manufacturer’s beta-lactam manufacturing equipment for the future production of non-beta-lactam compounds. Testing was conducted using chlorine dioxide gas at various concentrations and exposure times in an effort to achieve the pharmaceutical manufacturer’s required 3-log (99.9%) reduction of eight different beta-lactams on various surfaces. After a period of cycle development, multiple chlorine dioxide gas cycles at various concentrations and exposure lengths were shown effective in inactivating the eight beta-lactam compounds to a successful degree.