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Next-generation sequencing (NGS) enables parallel analysis of multiple genomic targets. The increasing demand for NGS-based multiplexed molecular diagnostics requires standardized protocols and recommendations to ensure reproducibility and accuracy of test results for routine clinical decision making. However, the lack of clinical NGS data from multi-laboratory tests and the absence of inter-laboratory comparisons have hampered the establishment of instructive clinical NGS standards. To fill the gap, we set up Proficiency Testing (PT) for inter-laboratory comparison, in which formalin-fixed paraffin-embedded specimens from eight lung and eight colon cancers were analyzed by 15 European molecular diagnostic laboratories on three different platforms using multiple target enrichment systems. We first performed platform, test, and informatics pipeline validation and conducted sensitivity and specificity analysis by random in silico down-sampling. We then implemented a multi-level filtering strategy based on performance tests of base substitution, replicate runs, and Sanger sequencing verified variants. We finally applied the filter criteria to the NGS data from the respective PT participants and obtained high inter-laboratory agreement. We demonstrated accuracy, scalability, and robustness of NGS by means of PT, serving as a benchmark for detecting clinically actionable molecular alterations in research and diagnostic laboratories. In conclusion, this study strongly highlights the importance of establishing standards for NGS-based testing, particularly when the test results impact on clinical decisions, and systematically provides data sets from multiple different labs to infer such standards.