Activating EGFR mutations are important genetic alterations that have strong therapeutic implications for non-small cell lung cancer(NSCLC) patients. However, the role of KRAS mutations in this process is still under evaluation. Here, we report on the feasibility of a large‑scale EGFR and KRAS mutation analysis in the daily routine of a single center. NSCLCs from 2,387patients were screened for EGFR and KRAS mutations from January 2010 to September 2015. Mutational analyses were performed in a single laboratory using single strand conformation polymorphism(SSCP)-Sanger sequencing and matrix‑assisted laser desorption ionization‑time of flight(MALDI‑TOF) on Sequenom platform for EGFR and pyrosequencing for KRAS. Activating EGFR mutations were found in 14.1% of all tumors, whereas KRAS mutations were found in 30.5% of all tumors. Direct sequencing showed analyzable cytological, small biopsy and surgical specimen percentages of 90.3, 90.9and98.1%, respectively, whereas the MALDI‑TOF platform showed analyzable cytological samples, small biopsies and surgical specimens percentages of 94.6, 95.7and96.9%, respectively. The mean analytical turnaround times(TAT) were 4and3days for direct sequencing and the MALDI‑TOF platform, respectively. Our results confirm that small biopsy or cytological samples can be used for reliable EGFR and KRAS mutation testing and indicate that adopting the MALDI‑TOF platform reduces the rate of missed samples among the samples. Moreover, the 3-day analytical TAT of the MALDI-TOF multi-target technique is appropriate for clinical management and reduces the overall treatment decision time.