With the increasing use of γ-irradiated containers made of multilayer polymeric flexible films for food and biopharmaceutical applications, the possible migration of degradation products of the polymers and their additives is becoming a topic of concern. This article aims at highly reliably identifying the degradation products generated after gamma irradiation and their origin to later on assess their potential harmfulness in single-use containers. In this study, GC–MS is used to identify by-products created by γ-irradiation of primary and secondary antioxidants usually present in polyolefin-based biotechnological single-use materials and to confirm identification relevancy based on the literature survey or standard when available. Degradation pathways are proposed to account for the formation of by-products identified during the study and to list intermediates and other by-products present in too small amounts to be detected and identified accurately in all extractable studies.