Immunogenic responses to biotherapeutics often lead to termination of their development because the resulting anti-drug-antibodies (ADA) can negatively impact pharmacology, safety, and efficacy. To mitigate ADA risks, in vitro risk assessment assays in non-clinical settings are essential to enhance safety and efficacy of protein-based therapeutics. This study aimed to develop and validate a human in vitro immunogenicity T cell proliferation assay. However, there is a lack of comprehensive guidelines for managing product-related factors such as endotoxin contamination, which can significantly influence assay sensitivity and accuracy. Our investigation of the impact of endotoxins revealed that levels above 0.1 EU/mg significantly induce T cell proliferation and CD14+ myeloid cell expansion, leading to potential false-positive outcomes in immunogenicity assessments. These findings suggest the importance of developing standardized protocols to enhance the predictive capability of in vitro methods, ensuring the assessment of therapeutic proteins accurately reflects their immunogenic potential without interference from contaminants.