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Lung cancer is one of the quickest and most fatal diseases and is divided into two types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Approximately 50% of NSCLC patients harbour mutations in the epidermal growth factor receptor (EGFR) gene, spanning from exon 18 to exon 21, which are responsible for the tyrosine kinase domain of the EGFR protein. Currently, gefitinib and erlotinib are two drugs inhibiting EGFR which are used in the treatment of lung cancer. The indication for use of these drugs depends on the mutation status of EGFR. Resistance to treatment with these two drugs has been found with mutations in exon 20; however, mutations in exon 18, 19, 21 benefited from the same treatment. Therefore, we built a molecular protocol for detecting mutations in exon 21 of EGFR in NSCLC patients in Vietnam which, in this study, is based on the realtime polymerase chain reaction high resolution melting (PCR HRM) technique. We successfully designed pairs of primers for the real-time PCR HRM technique to detect mutations in exon 21 and for the Sanger sequencing method to confirm the real-time PCR HRM results. We also investigated the optimal concentration of MgCl2 for the real-time PCR HRM protocol. Performance characteristics of the real-time PCR HRM protocol were evaluated in terms of specificity, limit of detection, repeatability, and it showed good results. Finally, we applied the molecular protocol to 172 clinical samples and compared these with the Sanger nucleotide sequencing method and the peptide nucleic acid (PNA) clamping method. The results of the comparison demonstrate that the molecular protocol constructed for detection of exon 21 mutations can be used in the treatment of NSCLC patients.