Immunoassays are prone to various interference effects that can compromise diagnostic accuracy, leading to false-positive or false-negative results. This chapter provides a comprehensive overview of common sources of interference—including cross-reactivities, nonspecific bindings, matrix effects, heterophilic antibodies, and human anti-animal antibodies—and their molecular mechanisms. It discusses how assay components, sample matrices, and labeling strategies contribute to these effects and outlines practical solutions such as optimized blockers, affinity-selective diluents, and antibody fragment use. Understanding and mitigating interference is essential for developing robust, reliable immunoassays across clinical, veterinary, and environmental applications.