Environmentally-sensitive membrane dyes have been extensively used to study the different liquid phases, (liquid-ordered (Lo) and liquid-disordered (Ld)) of the heterogenous cellular membrane. However, it is not yet well understood how these dyes affect membrane properties upon and post insertion. Using a combination of molecular dynamics (MD) simulations and fluorescence microscopy, we study the effect of PRODAN insertion upon its local environment. We firstly present the results of the MD simulations of PRODAN interacting with lipid bilayers of various compositions, specifically the resultant hydration and lipid order of the system. Experimentally, the lipid order of Lo and Ld vesicles containing various concentrations of PRODAN are inferred from their Generalised Polarisation (GP) values, calculated using their fluorescence spectra. We then apply the methodology to a more complex biological system, the HeLa cell line. For both systems, the presence of PRODAN influences its local environment differently between the Lo and Ld phases. In the simulated systems, the presence of PRODAN lowers the lipid order in the Ld phase and increases the order in the Lo phase, whilst experimental data demonstrates that even a small increase in PRODAN concentration significantly lowers the order of both phases. We suggest this discrepancy may be ascribed to the differing localisations of the dye molecules within the bilayer, and their effect on the hydration of adjacent lipids.