In the present study, excitation–emission matrix (EEM) fluorescence data of guest–host complexes between naproxen enantiomers and β-cyclodextrin were used to develop a second-order calibration method that was subsequently used to determine the enantiomeric composition of samples of naproxen. The chiral discrimination of naproxen enantiomers was realized via their difference in interaction with the chiral cavity of β-cyclodextrin due to their difference in stereochemical structure. The strategy combined the use of a self-weighted alternating normalized residue fitting (SWANRF) algorithm, for extraction of the pure analyte signal, with the standard addition strategy, for determination of naproxen enantiomers in the presence of a matrix effect caused by the proteins present in human urine. Feasible results were obtained in a molar fraction range from 60.0 to 85.0% of S-naproxen, providing absolute errors lowers than 7.50%. Finally, molecular modeling was performed to determine the chiral recognition on a molecular level, and the difference in the interaction energies and the patterns of molecular interactions were discussed. The results were in good agreement with experimental data.