Immunogenicity can have devastating consequences on the safety and efficacy of therapeutic proteins. Therefore, evaluating and mitigating the risk of product immunogenicity is critical for the development these products. This study, showed that Betaseron and Extavia, which are reported to be more immunogenic among IFNβ products in clinical usage, contain residual innate immune response modulating impurities (IIRMIs) capable of activating NF-κB and induced expression of inflammatory mediators. These IIRMIs were undetectable in Rebif or Avonex. The stimulatory effect was attributed solely to IIRMIs because it was evident in murine cells lacking the interferon receptor (IFNAR). The IIRMIs in Betaseron and Extavia triggered NF-κB activation in HEK-293 cells bearing TLR2 and TLR4 in MyD88 dependent manner. Importantly, the IIRMIs in Betaseron induced up-regulation of IL-6, IL-1β, and ccl5 in the skin of IFNAR knock out mice following subcutaneous administration. This indicates that trace level IIRMIs in Betaseron could contribute to the higher immunogenicity rates seen in clinics. Together these data suggest that cell based assays can reveal subtle but clinically relevant differences in IIRMIs following manufacturing changes or between products with the same active ingredients but different manufacturing processes. Appreciating these differences may inform immunogenicity risk assessments.