Molecular diagnostics in cancer pharmacogenomics is indispensable for making targeted therapy decisions especially in lung cancer. For routine clinical practice, the flexible testing platform and implemented quality system are important for failure rate and turnaround time (TAT) reduction. We established and validated the multiplex EGFR testing by MALDI-TOF MS according to ISO15189 regulation and CLIA recommendation in Taiwan. Totally 8,147 cases from Aug-2011 to Jul-2015 were assayed and statistical characteristics were reported. The intra-run precision of EGFR mutation frequency was CV 2.15% (L858R) and 2.77% (T790M); the inter-run precision was CV 3.50% (L858R) and 2.84% (T790M). Accuracy tests by consensus reference biomaterials showed 100% consistence with datasheet (public database). Both analytical sensitivity and specificity were 100% while taking Sanger sequencing as the gold-standard method for comparison. EGFR mutation frequency of peripheral blood mononuclear cell for reference range determination was 0.002 ± 0.016% (95% CI: 0.000-0.036) (L858R) and 0.292 ± 0.289% (95% CI: 0.000-0.871) (T790M). The average TAT was 4.5 working days and the failure rate was less than 0.1%. In conclusion, this study provides a comprehensive report of lung cancer EGFR mutation detection from platform establishment, method validation to clinical routine practice. It may be a reference model for molecular diagnostics in cancer pharmacogenomics.