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This study addresses this methodological bottleneck by combining controlled experimental comparisons with a comprehensive literature synthesis to identify how protocol decisions\u2014from sample preservation and DNA extraction to sequencing platforms and taxonomic assignments\u2014affect the results of COI metabarcoding and its interpretation. Overall biodiversity and community patterns were recovered by all combinations of tested methods, supporting the notion that patterns identified through DNA metabarcoding are robust and comparable across studies. We identify TES (Tris\u2010EDTA\u2010SDS) buffer, optionally paired with at\u2010sea homogenisation, as a practical alternative to ethanol preservation for large\u2010scale monitoring surveys. We show that integrating several classification methods and reference databases for taxonomic assignment improves diversity estimates and confidence in the assignments, and advocate for increased use of tools like BOLDigger that facilitate manual curation of ambiguous\/erroneous references. Finally, we demonstrate that introducing stricter filtering thresholds reduces the effect of false positives, pseudogenes and lab\u2010specific contamination, and make comparisons of data generated by different laboratories and methodological configurations more robust, although potentially at the expense of excluding rare taxa. While we intentionally refrain from recommending a universal best practices protocol, this study aims to provide a practical roadmap to help enhance the reliability and reproducibility of marine zooplankton monitoring via DNA metabarcoding.<\/jats:p>","DOI":"10.1111\/1755-0998.70149","type":"journal-article","created":{"date-parts":[[2026,5,22]],"date-time":"2026-05-22T11:02:02Z","timestamp":1779447722000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Optimising Zooplankton DNA Metabarcoding: Methodological Considerations for Large\u2010Scale Monitoring"],"prefix":"10.1111","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9007-2811","authenticated-orcid":false,"given":"Elizaveta","family":"Ershova\u2010Menze","sequence":"first","affiliation":[{"name":"Akvaplan\u2010Niva  Bergen Norway"},{"name":"Institute of Marine Research  Bergen Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jon\u2010Ivar","family":"Westgaard","sequence":"additional","affiliation":[{"name":"Institute of Marine Research  Bergen Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Helene","family":"Hjellnes","sequence":"additional","affiliation":[{"name":"Institute of Marine Research  Bergen Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tone","family":"Falkenhaug","sequence":"additional","affiliation":[{"name":"Institute of Marine Research  Bergen Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,5,22]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00227\u2010016\u20102920\u20100"},{"key":"e_1_2_11_3_1","unstructured":"Ahyong S. 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