A Visual Inspection Challenge Set for protein-based pharmaceutical products is a validated, regulatory-compliant tool used to assess, qualify, and maintain the performance of manual and automated inspection systems, particularly for sterile biologics such as monoclonal antibodies, recombinant proteins, and enzymes. These challenge sets are comprised of meticulously prepared and pre-classified test units containing a range of critical, major, and minor defects that simulate real-world anomalies encountered during fill-finish operations and aseptic processing. Common defects include visible protein aggregates, subvisible particulates, foreign matter (glass, fiber, rubber), container-closure integrity failures, and formulation-related issues such as turbidity or flocculation. The challenge set is utilized under controlled lighting and background conditions as per USP <790>, USP <1790>, and EU GMP Annex 1 guidelines to ensure consistent inspection sensitivity. Each defect sample is documented with traceable batch data, photographic evidence, and classification per ISO 2859-1 and ICH Q9 risk assessment principles. These sets are essential for inspector qualification, requalification, system validation, and training programs, supporting regulatory readiness for inspections by agencies such as the USFDA, EMA, and MHRA. A well-designed visual inspection challenge set enhances detection capability, ensures compliance, and strengthens quality assurance programs in biologic drug manufacturing.