In the past few years, we have witnessed a revolution in the molecular understanding of non-small cell lung cancer. Major progress has also been made in the clinic, with the introduction of EGFR-targeted and anti-angiogenic therapies. These advances have led to the development of a multitude of commercially available prognostic and predictive biomarkers. In particular, EGFR mutation and EML4/ALK testing have reached clinical validation and are incorporated into current treatment paradigms. This overview will present the scientific background of the biology of the relevant biomarkers and the studies conducted for their clinical validation. The technical challenges and shortcomings of these assays are also discussed. Furthermore, ongoing biomarker-driven clinical studies and the appropriate clinical use of available tests will be reviewed to assist the clinician with the proper incorporation of molecular testing into the routine care of patients with non-small cell lung cancer.