An enduring goal of personalized medicine in cancer is the ability to identify patients who are likely to respond to specific therapies. Our growing understanding of the biology and molecular signatures of individual tumor types has facilitated the identification of predictive biomarkers and has led to an increasing number of diagnostic tests to be performed, often as serial and distinct assays on limited tumor specimens. The biomarker diagnostics field has been revolutionized by next-generation sequencing (NGS), which provides a comprehensive overview of the genomic profile of a tumor. Many preanalytic variables can influence the accuracy and reliability of NGS results. Standardization of preanalytic variables is, however, complicated by the plethora of specimen acquisition and processing methods. Variables across the tissue journey, including specimen acquisition, specimen fixation, and sectioning, as well as postfixation processing, such as nucleic acid extraction, library preparation, and choice of sequencing methods, are critical for the reliability of NGS analysis; thus, standardization would be beneficial. In this article, each step in the tissue journey is outlined, with specific focus on preanalytic variables that can influence NGS results. Practical considerations for standardization of these variables are provided to facilitate accurate, reliable, and reproducible NGS-based molecular characterization of tumors, ultimately informing diagnosis and guiding treatment.