Context.—Technologic advances have contributed to the increasing relevance of RNA analysis in clinical oncology practice. The different genetic aberrations that can be screened with RNA include gene fusions and splice variants. Validated methods of identifying these alterations include fluorescence in situ hybridization, immunohistochemistry, reverse transcription–polymerase chain reaction, and next-generation sequencing, which can provide physicians valuable information on disease and treatment of cancer patients.Objective.—To discuss the standard techniques available and new approaches for the identification of gene fusions and splice variants in cancer, focusing on RNA analysis and how analytic methods have evolved in both tissue and liquid biopsies.Data Sources.—This is a narrative review based on PubMed searches and the authors' own experiences.Conclusions.—Reliable RNA-based testing in tissue and liquid biopsies can inform the diagnostic process and guide physicians toward the best treatment options. Next-generation sequencing methodologies permit simultaneous assessment of molecular alterations and increase the number of treatment options available for cancer patients.