Knowledge of how an in vitro aerosol exposure system delivers a test aerosols to the biological test system is among the most crucial prerequisites for the interpretation of exposure experiments and relies on detailed exposure system characterization. Although various methods for this purpose exist, many of them are time consuming, require extensive instrumentation, or offer only limited ability to assess the performance of the system under experimental settings. We present the development and evaluation of a new, highly robust and sensitive fluorometry-based method for assessing the particle size specific delivery of liquid aerosols. Glycerol aerosols of different mean particle sizes and narrow size distributions, carrying the fluorophore disodium fluorescein, were generated in a condensation monodisperse aerosol generator. Their detailed characterization confirmed their stability and the robustness and reproducibility of their generation. Test exposures under relevant experimental settings in the Vitrocell® 24⁄48 aerosol exposure system further confirmed their feasibility for simulating exposures and the high sensitivity of the method. Potential applications of the presented method range from the experimental confirmation of computationally simulated particle dynamics, over the characterization of in vitro aerosol exposure systems, to the detailed description of aerosol delivery in test systems of high complexity.