A fluorimetric study on the spectral characteristics of two antileukotrienes, cinalukast and montelukast, has been performed. Ionization constants of both of them have been photometrically calculated. Cinalukast pKa in ethanol:water 50:50 (v/v) medium resulted to be 2.2 ± 0.1. Because the spectral characteristics of montelukast are widely affected by the solvent nature, pKa was estimated in two different ethanol:water media, 70:30 (v/v) and 10:90 (v/v) and the values calculated were pKa = 2.9 ± 0.1, and pKa1 = 2.0 ± 0.1 and pKa2 = 6.5 ± 0.1, respectively. It has been proven that the fluorescence of both, cinalukast and montelukast, is significantly intensified in the presence of cyclodextrins (CyDs). The host–guest complexation processes between cinalukast and α-CyD or heptakis-(2,6-di-O-methyl)-β-cyclodextrin (DIMEB) and between montelukast and DIMEB have been investigated by fluorescence spectroscopy. A 1:1 stoichiometric ratio was established for the three studied inclusion complexes. The changes produced on the fluorescence of cinalukast or montelukast, when they are included on the hydrophobic CyD cavity are used to calculate their association constants by a non-linear regression method. Semiempirical MO calculations using AM1 method were performed in order to characterize the studied inclusion complexes. A new method for cinalukast determination in human serum, based on the fluorescence of the complex cinalukast–DIMEB exhibiting limit of detection of 7.95 ng mL−1 has been proposed with satisfactory results. Adequate recovery values between 95 and 103% were calculated at five different concentration levels.