OBJECTIVE To identify issues during donning and doffing of personal protective equipment (PPE) for infectious diseases and to inform PPE procurement criteria and design. DESIGN A mixed methods approach was used. Usability testing assessed the appropriateness, potential for errors, and ease of use of various combinations of PPE. A qualitative constructivist approach was used to analyze participant feedback. SETTING Four academic health sciences centers: 2 adult hospitals, 1 trauma center, and 1 pediatric hospital, in Toronto, Canada. PARTICIPANTS Participants (n=82) were representative of the potential users of PPE within Western healthcare institutions. RESULTS None of the tested combinations provided a complete solution for PPE. Environmental factors, such as anteroom layout, and the design of protocols and instructional material were also found to impact safety. The study identified the need to design PPE as a complete system, rather than mixing and matching components. CONCLUSIONS Healthcare institutions are encouraged to use human factors methods to identify risk and failure points with the usage of their selected PPE, and to modify on the basis of iterative evaluations with representative end users. Manufacturers of PPE should consider usability when designing the next generation of PPE. Infect Control Hosp Epidemiol 2016;37:1022-1028.