Abstract. Fluorescent dyed polystyrene latex spheres (PSLs) are commonly used for characterization and calibration of instruments detecting fluorescence signals from particles suspended in the air and other fluids. Instruments like the Ultraviolet Aerodynamic Particle Sizer (UV-APS) and the Waveband Integrated Bioaerosol Sensor (WIBS) are widely used for bioaerosol research, but these instruments present significant technical and physical challenges requiring careful characterization with standard particles. Many other research communities use flow cytometry and other instruments that interrogate fluorescence from individual particles, and these also frequently rely on fluorescent PSLs as standards. Nevertheless, information about physical properties of commercially available PSLs provided by each manufacturer is generally proprietary and rarely available, making their use in fluorescence validation and calibration very difficult. This technical note presents an overview of steady-state fluorescence properties of fluorescent and non-fluorescent PSLs, as well as of polystyrene-divinylbenzene (PS-DVB) particles, by using on- and offline spectroscopic techniques. We show that the “fluorescence landscape” of PSLs is more complex than the information typically provided by manufacturers may imply, especially revealing multimodal emission patterns. Furthermore, non-fluorescent PSLs also exhibit defined patterns of fluorescent emission originating from a mixture of polystyrene and detergents, which becomes a crucial point for fluorescence threshold calibrations and qualitative comparison between instruments. By comparing PSLs of different sizes, but doped with the same dye, changes in emission spectra from bulk solutions are not immediately obvious. On a single-particle scale, however, fluorescence intensity values increase with increasing particle size. No significant effect in the fluorescence signatures was detectable by comparing PSLs in dry vs. wet states, indicating that solvent water may only play a minor role as a fluorescence quencher. Because information provided by manufacturers of commercially available PSLs is generally very limited, we provide the steady-state excitation–emission matrices (EEMs) of PSLs as open-access data within the Supplement. Detergent and solvent effects are also discussed in order to provide information not available elsewhere to researchers in the bioaerosol and other research communities. These data are not meant to serve as a fundamental library of PSL properties because of the variability of fluorescent properties between batches and as a function of particle aging and agglomeration. The data presented, however, provide a summary of spectral features which are consistent across these widely used fluorescent standards. Using these concepts, further checks will likely be required by individual researchers using specific lots of standards.