Genes and their products involved in the biological pathways of human cancers have been studied as either targets of new therapies, or predictive markers for the sensitivity of or resistance to the therapies. Companion diagnostic testing on biological markers for targeted cancer therapies has become a vital component of personalized cancer treatment. This article provides an overview on the biological pathways and biomarkers, including EGFR, KRAS, BRAF, ALK, ROS1, HER2, and KIT for targeted treatment of lung, gastrointestinal, colorectal, and breast cancers as well as malignant melanoma. The current testing approach appears to focus on single biomarkers. However, a comprehensive approach that includes testing multimarkers involved in the mitogen-activated protein kinase, phosphoinositide 3-kinase/protein kinase B, and mammalian target of rapamycin pathways may become more desirable for some cancers, because of therapy resistance that can be caused by mutations in different genes and the availability of new therapies that may aim at multiple targets in the pathways. Only a few companion diagnostic kits have been approved by FDA, and the use of an FDA approved kit for some biomarkers, such as BRAF and KRAS, can be controversial.