Astemizole (AST) is non-fluorescent in aqueous solution. This property makes its determination through direct fluorescence methods difficult. Reaction and supramolecular interaction mechanisms, between AST and palmatine (PAL) as they compete for occupancy of the cucurbit[7]uril (CB[7]) cavity, were studied using spectrofluorimetry, ¹H NMR, and molecular modeling calculations. The association constants of the complexes formed between the host and the guest were determined. Based on the significant quenching of the supramolecular complex fluorescence intensity, a fluorescent probe method of high sensitivity and selectivity was developed to determine AST in its pharmaceutical dosage forms and in urine samples with good precision and accuracy. The linear range of the method was from 0.02 μg mL⁻¹ to 2.2 μg mL⁻¹. The detection limit was 0.007 μg mL⁻¹. This shows that the proposed method has promising potential for therapeutic monitoring and pharmacokinetics and for clinical application.