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Abstract Targeted panel-based tumor mutation burden (TMB) assays are widely employed to guide immunotherapy for patients with solid tumors. However, the accuracy and consistency of this method can be compromised due to the variability in technical details across different laboratories, particularly in terms of panel size, somatic mutation detection and TMB calculation rules. Currently, systematic evaluations of the impact of these technical factors on existing assays and best practice recommendations remain lacking. We assessed the performance of 50 participating panel-based TMB assays involving 38 unique methods using cell line samples. In silico experiments utilizing TCGA MC3 datasets were performed to further dissect the impact of technical factors. Here we show that the panel sizes beyond 1.04 Mb and 389 genes are necessary for the basic discrete accuracy, as determined by over 40,000 synthetic panels. The somatic mutation detection should maintain a reciprocal gap of recall and precision less than 0.179 for reliable psTMB calculation results. The inclusion of synonymous, nonsense and hotspot mutations could enhance the accuracy of panel-based TMB assay. A 5% variant allele frequency cut-off is suitable for TMB assays using tumor samples with at least 20% tumor purity. In conclusion, this multicenter study elucidates the major technical factors as sources of variability in panel-based TMB assays and proposed comprehensive recommendations for the enhancement of accuracy and consistency. These findings will assist clinical laboratories in optimizing the methodological details through bioinformatic experiments to enhance the reliability of panel-based methods.