The biotherapeutic type I interferons (IFN-I) are indicated to treat several diseases. These products are regulated to guarantee safety and efficacy through critical quality attributes. For this purpose, the development of robust assays is required, followed by its validation to demonstrate their suitability for its intended purpose. Despite there are some commercial kits to evaluate IFN-I signaling, these are focused on measuring in vitro biological response instead of their validation, which is a pharmaceutical industry requirement. The aim of this work was to validate the HEK-Blue IFN-α/β system evaluating the biological activity of IFN-α/β under good laboratory practices, according to international standards. Our results demonstrated that HEK-Blue IFN-α/β system comply with accuracy (r2>0.95) precision (CV < 20%) and specificity for both IFN-α/β; confirming that this assay is robust for this biotherapeutics’ evaluation. Thereby, this bioassay could be implemented as a complementary method to the classical anti-proliferative and anti-viral assays under quality control environments.