There is worldwide concern with regard to the adverse effects of drug usage. However, contaminants can gain entry into a drug manufacturing process stream from several sources such as personnel, poor facility design, incoming ventilation air, machinery and other equipment for production, etc. In this validation study, we aimed to determine the impact and evaluate the contamination control in the preparation areas of the rapid transfer port (RTP) chamber during the pharmaceutical manufacturing processes. The RTP chamber is normally tested for airflow velocity, particle counts, pressure decay of leakage, and sterility. The air flow balance of the RTP chamber is affected by the airflow quantity and the height above the platform. It is relatively easy to evaluate the RTP chamber′s leakage by the pressure decay, where the system is charged with the air, closed, and the decay of pressure is measured by the time period. We conducted the determination of a vaporized H 2O 2 of a sufficient concentration to complete decontamination. The performance of the RTP chamber will improve safety and can be completely tested at an ISO Class 5 environment.