RTgill-W1 gill cells of the rainbow trout (Oncorhynchus mykiss) were subjected to supernatants from exponentially grown Alexandrium pseudogonyaulax cultures for 24 h to create dose-response curves. The bioassays were carried out with 300 µl of total incubation volume in 96-well plates. After 24 hours, 50 µl subsamples were transferred to a new 96-well plate for the assessment of lytic activity using a lactate dehydrogenase (LDH) cytotoxicity assay kit (CyQUANT™, Thermo Fisher Scientific, C20301) following the standard protocol provided by the manufacturer. The bioassays were analysed fluorometrically using a cell-imaging multi-mode reader (Cytation™ 3 Cell Imaging Multi-Mode Reader, BioTek). All data was pre-filtered according to (1) visual inspection of the gill cell densities in the 96-well plates and if densities differed greatly data was excluded, and (2) data were grouped by each biological gill cell line replicate, each biological A. pseudogonyaulax replicate, each strain and each cell density equivalent and subjected to a Dixon outlier test. If the Dixon test was significant, data was excluded. This dataset contains four to five biological gill cell line replicates tested with three different A. pseudogonyaulax strains, each comprised of up to three biological replicates. Each biological replicate of each A. pseudogonyaulax strain was tested with a total of three to four cell density equivalents of cell-free supernatants, each comprised of two to six technical replicates. The bioassays were carried out between May 19 and October 10, 2023 at the Department of Food Chemistry and Toxicology (LMC), Faculty of Chemistry, University of Vienna.