This work details a study whereby the characterization of chiral selectors and identification of optimal separation conditions is evaluated by steady-state fluorescence anisotropy measurements. Earlier studies in our laboratory have shown fluorescence anisotropy to be an effective tool in evaluating chiral recognition, and in this study, the feasibility of characterizing chiral separation systems by the technique is evaluated. Four chiral selectors were examined under various conditions to explore correlation between chiral separation ability and differences in the steady-state fluorescence anisotropy of the enantiomers measured under similar conditions. A good correlation between the fluorescence anisotropy data and separation data was observed with R2 values ranging from 0.9279 to 0.9959. The fluorescence anisotropy measurements were examined under conditions that mimicked chiral separation conditions and the feasibility of a priori optimization of chiral separations is discussed.