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11 pages, 5 figures, 2 tables, Phagotrophic mixotrophs (mixoplankton) are now widely recognised as important members of food webs, but their role in the functioning of food webs is not yet fully understood. This is due to the lack of a well-established technique to estimate mixotrophic grazing. An immediate step in this direction would be the development of a method that separates mixotrophic from heterotrophic grazing that can be routinely incorporated into the common techniques used to measure microplankton herbivory (e.g., the dilution technique). This idea was explored by the addition of rotenone, an inhibitor of the respiratory electron chain that has been widely used to selectively eliminate metazoans, both in the field and in the laboratory. Accordingly, rotenone was added to auto-, mixo-, and heterotrophic protist cultures in increasing concentrations (ca. 24 h). The results showed that mixotrophs survived better than heterotrophs at low concentrations of rotenone. Nevertheless, their predation was more affected, rendering rotenone unusable as a heterotrophic grazing deterrent. Additionally, it was found that rotenone had a differential effect depending on the growth phase of an autotrophic culture. Altogether, these results suggest that previous uses of rotenone in the field may have disrupted the planktonic food web, This project is a contribution of the Marine Zooplankton Ecology Group from the Generalitat de Catalunya (2017 SGR 87) and has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 766327, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)