The clinical reliability of liquid biopsy is critically determined by pre-analytical variables rather than analytical technologies alone. Blood is a highly dynamic biological environment, and analyte composition can change rapidly after collection. Variables such as collection tubes, anticoagulants, centrifugation protocols, storage conditions, and processing delays directly affect assay sensitivity and specificity. CTCs, ctDNA, and EVs exhibit distinct stability profiles, making a single standardized protocol insufficient for all analytes. In multicenter studies, discrepancies in performance are often attributable to subtle differences in pre-analytical handling. International societies have proposed minimum standards, but universal harmonization remains challenging. Clinical workflows frequently require compromises between technical rigor and practical feasibility. Pre-analytical processing should therefore be regarded as a core determinant of clinical reproducibility. This chapter systematically examines analyte-specific pre-analytical variables and standardization efforts.