Although H2O2 decontamination is an established process in the pharmaceutical industry, there are many theoretical and practical aspects that need to be considered. Some of them are well known and obvious but are not taken into account in the development of modern isolators, others are highly dependent on the knowledge, experience and practical skills of the team performing the qualification and daily use. Consideration of both aspects is crucial for successful H2O2 decontamination. By fully understanding the theoretical and practical aspects, isolators can be designed and operated to ensure reliable and effective decontamination.