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This is a laboratory study to develop protocols for the validation of ethylene oxide sterilization. This study includes methodologies for physical, microbiological, and residual chemical qualification using a validated gas chromatography technique (GC/FID). The present study allowed for the optimization of the sterilization process, ensuring safety, robustness, and increased process efficiency by reducing the residual aeration time from 48 hours to 6 hours. The innovative methodologies of this research incorporated the use of physical sensors and chemical integrators, as well as the use of sterility tests and endotoxin assays in comparison to the biological monitoring technology of the rapid bioindicator test. Quality control analyses, qualifications, and validation of all stages of sterilization ensured the effectiveness of the process, in accordance with the criteria of the Brazilian Health Regulatory Agency, allowing for productivity gains and increased system performance.