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The production of reactive oxygen species (ROS) is an unavoidable consequence of oxygenic photosynthesis and represents a major cause of oxidative stress in phototrophs, having detrimental effects on the photosynthetic apparatus, limiting cell growth, and productivity. Several methods have been developed for the quantification of cellular ROS, however, most are invasive, requiring the destruction of the sample. Here, we present a new methodology that allows the concurrent quantification of ROS and photosynthetic activity, using the fluorochrome dichlorofluorescein (DCF) and in vivo chlorophyll a fluorescence, respectively. Both types of fluorescence were measured using an imaging Pulse Amplitude Modulation (PAM) fluorometer, modified by adding a UVA-excitation light source (385 nm) and a green bandpass emission filter (530 nm) to enable the sequential capture of red chlorophyll fluorescence and green DCF fluorescence in the same sample. The method was established on Phaeodactylum tricornutum Bohlin, an important marine model diatom species, by determining protocol conditions that permitted the detection of ROS without impacting photosynthetic activity. The utility of the method was validated by quantifying the effects of two herbicides (DCMU and methyl viologen) on the photosynthetic activity and ROS production in P. tricornutum and of light acclimation state in Navicula cf. recens Lange-Bertalot, a common benthic diatom. The developed method is rapid and non-destructive, allowing for the high-throughput screening of multiple samples over time., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)