Abstract: Objective :To establish a research model for screening seven different genotypes of codon 12 in K-ras alleles and to discuss its clinical application. Methods :In this assay, the man-made plasmid DNAs were used as the templates. First specific DNA fragments containing the target site (s) were amplified from the plasmid DNA, then through sample dephosphorylation, and two ordinary oligodeoxyribonucleotide primers without any modification were used, followed by the primer extension reaction in the presence of dATP, ddTTP, ddCTP and ddGTP mixture, the products were then analyzed using MALDI-TOF mass spectrometry by detecting the m/z ratio. According to the peak value in the mass spectrogram the genotypes were analyzed. Results :The unambiguous result could be achieved through MALDI-TOF-MS coupled with primer extension method and 384 samples could be analyzed simultaneously. Conclusions :MALDI-TOF-MS coupled with primer extension method is accurate and stable, and the reaction system takes the advantage of automation and high-throughput. It is likely to be an alternative approach in early diagnosis and cancer screening in future.