This dataset illustrates the Best Practices in flow cytometry for studying phytoplankton. The use of flow cytometry to collect datasets on phytoplankton functional groups is rapidly expanding worldwide, with deployments on scientific vessels, ships of opportunity, buoys, and autonomous environmental monitoring platforms. Automated flow cytometry for aquatic photosynthetic microbes enables precise quantification across the full-size range of phytoplankton while offering enhanced autonomy. Sharing these datasets with the scientific community—whether to improve the resolution of global phytoplankton distribution or to facilitate the intercomparison of environmental indicators among monitoring laboratories—requires quality-controlled instruments and standardized data acquisition. Although flow cytometers all operate on the principle of recording the optical pulse shapes of particles passing through a laser beam, their configurations vary in terms of laser wavelength and power, sheath fluid management, sample inlet, and dataset output format. The characterization of phytoplankton communities and their optical representation on cytograms has been standardized by experts, with resolution depending on the optimal configuration of the instrument in use. This dataset exemplifies the application of best practices for optimizing the settings of autonomous pulse shape-recording flow cytometers, such as those developed by CytoBuoy. We address key aspects such as particle counting limits, the coincidence phenomenon, trigger threshold optimization, and regular quality control procedures, illustrating these principles with datasets from two types of instruments. The primary goal is to establish a methodological framework that guides and supports the exploration and application of this type of flow cytometer, ultimately achieving reliable and optimal sample acquisition resolution.