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Early detection of invasive species is crucial to deal effectively with biological invasions in ports, which are hotspots of species introductions. In this study, a simplified end-time PCR methodology conducted on eDNA from water samples was developed for rapid detection of the invasive seaweed Asparagopsis armata (four hours from water collection to result visualization). It was tested dockside in four international Spanish ports in presence of stakeholders, whose feedback was obtained to explore the real applicability of this biotechnology. Although biological invasions were not a main concern for them, results indicate a unanimous approval of the methodology by the stakeholders, having detected the presence of A. armata in three of the ports. Stakeholders suggested further developments for easier application of the tool and multiple species detection, to be adopted for the control of invasive species in ports. • Ports are hotspots for the introduction of new species. • Biological invasions are most effectively treated with early detection. • A fast PCR protocol to detect species eDNA from water samples was developed. • Invasive alga Asparagopsis armata was found from three ports in <4 h. • Stakeholders evaluated positively this tool for rapid species detection. [ABSTRACT FROM AUTHOR]