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The Biofluid Biomarkers Best Practices Workgroup of the National Alzheimer's Coordinating Center-Alzheimer's Disease Research Center (ADRC) Biomarker Core Steering Committee was convened to update pre-analytical handling guidelines for biofluid biomarkers, focusing on cerebrospinal fluid (CSF) and blood. We reviewed current literature pertinent to best practices for biomarker studies and surveyed the ADRCs for biomarker analytes, platforms, and protocols used at each center. Across 37 ADRCs, 16 CSF and 28 plasma/serum analytes were reported to be studied at multiple centers. The pre-analytical handling steps and concerns related to each, as supported by empirical studies and expert opinion, were integrated to generate a revised guideline document. The guideline aimed to standardize steps in biospecimen and biomarker analyte collection, storage, and pre-analytical handling across the ADRCs. The 2025 ADRC guidelines represent the current working knowledge on biomarker best practices, providing guidance and harmonized protocols, and promoting robust analysis and reporting of composite data.