The inclusion complexes of Acetanilide (AA) with 18-Crown-6 (18C6) were studied by UV and fluorescence techniques. The absorption and emission of AA in 18C6 medium confirms the –NH-CO-CH3 aromatic side chain AA is inserted into the 18C6 cavity. The stoichiometry and binding constant of 1:1 inclusion complex was calculated using the Benesi–Hildebrand plot derived UV and fluorescence studies and the thermodynamic parameters (ΔG) of inclusion process were also determined. The results indicated that the inclusion process was an exergonic and spontaneous process. The complex was investigated by semiempirical and molecular docking methods. The AA: 18C6 inclusion complex obtained by molecular docking studies was in good correlation with the results obtained through experimental methods.