Background: Next-generation sequencing (NGS) testing has two analytical processes, wet bench and bioinformatics process. Exome sequencing covers about 20000 human protein-coding gene sequences. Since these sequences are only 2% of human genome, but can predict 85% of human gene related diseases, whole exome sequencing is the most cost-effective test to diagnose unknown genetic diseases. Methods: Analysis methods developed by the Department of Laboratory Medicine of China Medical University Hospital (CMUH) with compliance of the molecular pathology checklists of the College of American Pathologists (CAP). Results: We developed the exome sequencing analysis workflow. First, single nucleotide polymorphism, SNP known with a minor allele frequency (MAF) >1%, was excluded. Second, variants other than SNP detected by NGS are submitted to the ClinVar database, which divided the relationships between variants and clinical significances into five categories: benign, likely benign, uncertain, likely pathogenic, and pathogenic. Third, all pathogenic variants, also confirmed by Sanger sequencing, might be current clinical relevance or incidental findings. Fourth, uncertain clinical significance variants with a MAF 30% were underwent further analysis by three pathogenicity predictions software: SIFT, PolyPhen, and CADD_PHRED. The final report from of exome sequencing contain sections of summary, clinical relevance (pathogenic), incidental finding (pathogenic), benign / likely benign, GWAS-related diseases, uncertain significance (including the results of three pathogenic software analysis), and the performance of the NGS platform. Conclusions: we demonstrated a reasonable working flow and a clinical practicable reporting format of exome sequencing by NGS.