Fluorescence solvent relaxation experiments are based on the characterization of time-dependent shift in the fluorescence emission of a chromophore, yielding polarity and viscosity information of the chromophore immediate environment. Applied to phospholipid bilayers a defined location of the chromophore with respect to the z-axis of the bilayer allows monitoring of hydration and mobility of the probed segment of the lipid molecules. Specifically, time-resolved fluorescence experiments, fluorescence quenching data and molecular dynamic (MD) simulations show that 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) reports on hydration and mobility of the sn-1 acyl groups in a phosphatidylcholine bilayer. The time-dependent fluorescence shift (TDFS) of Laurdan reports on headgroup compression and expansion induced by the addition of different amounts of cationic lipids to phosphatidylcholine bilayers, which was predicted by previous MD simulations. Addition of truncated oxidized phospholipids leads to increased mobility and hydration at the sn-1 acyl level. That experimental finding can be explained by MD simulations, indicating that the truncated chains of the oxidized lipid molecules are looping back into aqueous phase, hence creating voids below the glycerol level. Fluorescence solvent relaxation experiments are also useful for the understanding of salt effects on the structure and dynamics of lipid bilayers. Those experiments for example demonstrate that large anions increase hydration and mobility at the sn-1 acyl level of phosphatidylcholine bilayers, which could not be explained by standard MD simulations. However, when introducing polarizability into the applied force field, those simulations show that big soft polarizable anions are able to interact with hydrophilic/hydrophobic interface of the lipid bilayer penetrating to the level probed by Laurdan and that they expand and destabilize the bilayer making it more hydrated and mobile.