Decontamination with hydrogen peroxide is a technology widely used to reduce microbial contamination. A typical application of this technology is in the decontamination of sterility test isolators. This article describes how to decontaminate sterility test isolators and validate the process in order to demonstrate that the microbiological target has been achieved and that the risk of false negatives due to residuals of hydrogen peroxide is excluded. Hydrogen peroxide can adversely affect some materials, resulting in inhibition of microbial growth. A package integrity verification, focused on the risk of penetration of decontaminating agent into different product containers and through different materials, is one of the main topics. Several case studies let readers understand the most critical items, choose their materials correctly, and validate the process itself. Hydrogen peroxide measurements on the surface of several materials, inside the primary packaging container, and inside aqueous solutions are part of this article. Validation of the decontamination cycle as well as validation of the operative procedure are key elements for a good laboratory practices approach.