Recent sequencing efforts in melanoma have elucidated many previously unknown molecular pathways and biological mechanisms involved in melanoma development and progression and have yielded a number of promising targets for molecular therapy. As sequencing technologies have become more sophisticated and have revealed an ever-increasing complexity of the genetic landscape of melanoma, it has become clear that sequencing methods applied to clinical specimens have to reliably capture not only recurrent “hotspot” mutations like BRAFV600 and NRASQ61 or “mini-hotspot” mutations like exon 11 and 13 c-KIT but also heterogeneous somatic mutations dispersed across multiple functionally conserved regions of genes or entire genes. One such example in melanoma is the ERBB4 receptor, or HER4, a member of the Erb receptor family, which has recently been shown to be a major oncogenic “driver” in melanoma. Mutated ERBB4 signaling activates both aberrant ERBB4 and PI3K-AKT signal transduction, mediates sensitivity to small-molecule inhibition with the dual-tyrosine kinase inhibitor lapatinib, and has recently also been implied in oncogenic glutamatergic signaling in melanoma. Mutations involving the ERBB4 gene act as “gain-of-function” mutations and predominantly involve the extracellular domains of the receptor. Additional sequencing efforts have recently identified recurrent mutations (“mini-hotspots”) or mutation clusters which affect the regulation of, e.g., ligand binding, arrangement of extracellular domain alignment, or intramolecular tether formation. In this chapter, we describe the methods used to determine the mutation status of all exons of the ERBB4 gene in clinical specimens obtained from patients afflicted by metastatic melanoma. Upon slight modifications, this protocol can also be used for mutational analysis of other oncogenes affected by “non-hotspot” mutations dispersed across multiple exons. This sequencing technique has successfully been applied within a clinical trial selecting patients with ERBB4-mutant melanoma for lapatinib treatment. With the increasing emergence of low-frequency oncogenes affected by heterogeneous activating mutations located in different exons and regions this method will provide a mean to translate the promise of recently obtained genetic knowledge into clinical genotype-directed targeted therapy trials.