<p>The development and implementation of environmental DNA (eDNA) assays have revolutionized aquatic species monitoring, yet progress is impeded by inconsistent standards in assay design, validation, and reporting. When insufficiently validated quantitative PCR (qPCR) assays are used for management and conservation, false positives or false negatives can have significant ecological and policy consequences. Drawing on existing standardized workflows, we present an integrated, multi-stage decision-oriented framework for eDNA assay development and validation that spans conceptual design through to in situ testing. The first stage includes a decision checklist for in silico assay validation, adapted from the empirical Thalinger validation scale, which enables users to critically assess the risk of false positives and false negatives before entering the laboratory. This early-stage evaluation informs and strengthens subsequent in vitro (i.e., specificity, sensitivity, and interference) and in situ testing, the key principles of which are presented here in a streamlined and accessible manner to support both expert and non-expert users. We illustrate the flexibility and scalability of these guidelines through three diverse case studies, from regional validation of a published assay to design and validation of novel genus-specific and species-specific assays for application to water samples and dietary analysis, respectively. Our trainee-led initiative reflects interdisciplinary partnerships across academic institutions, government agencies, and conservation organizations, and demonstrates how co-produced molecular tools can generate actionable results for fisheries management. The resulting decision support tool offers a reproducible, transparent, and adaptable pipeline for assay development, with broad applicability across freshwater fish monitoring programs in Canada and beyond.</p>