Non-small cell lung cancer (NSCLC) is one of the most common cancers worldwide. An estimated 170,000 new diagnoses are expected this year. Recently, small molecule inhibitors directed at the EGFR kinase domain were approved for the treatment of advanced stages of NSCLC. Genotyping of the EGFR kinase domain has proven to be a useful marker for predicting who will respond to these novel medications. This unit provides a protocol to perform mutation analysis on the EGFR kinase domain where mutations have been associated with significant responsiveness to these EGFR inhibitors. The protocol includes microdissection of tumor tissue from slides, DNA digestion of these cells, amplifying and sequencing pertinent segments of the EGFR gene, and interpretation of the data. The protocol is designed with appropriate redundancy to eliminate allele dropout and to maximize detection of somatic mutations within the tumor.