Here we use the excitation generalized polarization (GP(ex)) of 6-lauroyl-2-(dimethylamino)naphthalene (Laurdan) fluorescence in fluid cholesterol/1-palmitoyl-2-oleoyl-L-alpha-phosphatidylcholine large unilamellar vesicles to explore the experimental conditions that would be required in order to detect a biphasic change in membrane properties at specific sterol mole fractions (C-r) (e.g., 20.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol %) for maximal sterol superlattice formation. Laurdan's GP(ex) changes with sterol content in an alternating manner, showing minima (termed as GP(ex) dips) at similar to C-r. GP(ex) dips are detectable if the vesicles are preincubated for a sufficient time period and protected from sterol oxidation. In most cases, vesicles with a higher lipid concentration take a longer time to show a GP(ex) dip at C-r. The depth of the GP(ex) dip increases with increasing incubation time and eventually reaches a plateau, at which the maximum area covered by superlattices is expected to be achieved. However, if the vesicles are not protected against sterol oxidation, the GP(ex) dips are attenuated or obliterated. These effects can be attributed to the increased inter-bilayer lipid exchange and the increased vesicle-vesicle interactions present at high lipid (vesicle) concentrations as well as the decreased interactions between oxysterols and phospholipids. These possible explanations have been incorporated into a kinetic model that is able to calculate the effects of sterol oxidation and lipid concentration on the depth of the GP(ex) dip. The depth of the GP(ex) dip, the required incubation time for the dip formation, and the lipid concentration dependence of the GP(ex) dip vary with C-r, suggesting different physical properties for different sterol superlattices. To detect a biphasic change in membrane properties at C-r, one should also use small sterol mole fraction increments over a wide range, keep all of the vesicles in the same sample set under the same thermal history, and consider lipid concentration, probe type, and C-r value. These results improve our mechanistic understanding of sterol superlattice formation and explain why some studies, especially those requiring high lipid concentrations, did not detect a biphasic change in membrane properties at C-r.