Background: The ability to rapidly, inexpensively, and accurately identify cancer patients based on actionable genomic mutations in tumour specimens is becoming critically important in routine clinical diagnostics. Targeted panel sequencing is becoming popular because it enables comprehensive and cost-effective diagnosis. However, the implementation of a next-generation sequencing (NGS) assay in clinical settings requires careful analytical validation to demonstrate its ability to detect multiple genomic variants. Materials and Methods: Here, we developed a custom-targeted NGS panel to identify actionable variants, including single nucleotide variants, insertions, and deletions; copy number variants; and gene fusions, across 73 genes for targeted cancer therapy. We implemented a practical validation strategy with diluted samples and reference standard samples that modelled key determinants of accuracy, including mutant allele frequency, insertion/deletion length, amplitude of copy number, and hotspot gene fusions. Results: The analytical validation results demonstrated that our panel can identify different types of genomic alterations in these test samples with high levels of accuracy, sensitivity, and reproducibility. Conclusion: Our panel could be deployed as a routine clinical test to comprehensively detect actionable variants in cancer patients to guide targeted therapy decisions.