6-p-Toluidinylnaphthalene-2-sulfonate (TNS) is used as a fluorescent probe for exploring hydrophobic regions of several biological substances, such as proteins, and studying solution state folding behaviour. The current study examines the complexation of TNS with 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD) in aqueous solution, mainly by ultraviolet spectrophotometry using various concentrations of HPbetaCD. The structure of HPbetaCD was confirmed by using positive-ion electrospray ionization (ESI+) mass spectrometry. The complex was examined for its stoichiometry applying the continuous variation (Job plot) method. Also, the kinetics of the complex formation was monitored and the determination of the stability constant was calculated. For this purpose, the spectrophotometric properties of TNS were observed in the presence of increasing concentrations of HPbetaCD applying the transformed Benesi-Hildebrand linear model as well as a nonlinear one. The results suggest that TNS forms a stable complex of 11 molar ratio, at least at the examined concentrations. Furthermore, from the measurements of 1H nuclear magnetic resonance (1H NMR) spectra, interactions between protons of TNS with HPbetaCD were determined.