Fluorescence spectroscopy is one of the most common methods of analyzing atmospheric aerosols and cloud water making it of fundamental interest to understand the molecular origin of the underlying fluorophores. In this work, experimental characterization of the spectroscopic properties of aqueous aerosol/cloud water mimics containing glyoxal and ammonium sulfate precursors is presented. Spectroscopic results include mass absorption coefficients, excitation–emission matrix (EEM) spectra, fluorescence quantum yields, and fluorescence lifetimes. The chemical composition of these samples is also investigated using electrospray ionization mass spectrometry. The synthesized samples were irradiated with a solar simulator to study changes in the spectroscopic properties and estimate the atmospheric lifetimes with respect to direct photolysis and OH oxidation. The EEM spectra of the mimics closely resemble protein-like fluorescence due to the emission of biimidazole with an estimated fluorescence quantum yield of ϕ = 0.82 ± 0.05 at λex = 275 nm. Biimidazole and tryptophan fluorescence are explored in detail with EEM and time-resolved fluorescence (TRF) spectroscopy to compare the two fluorophores. The results indicate that biimidazole can be mistaken for tryptophan-containing proteins in atmospheric samples, but fluorescence lifetime measurements can potentially distinguish the two fluorophores.