Abstract In this article, we investigate the effect of guanidinium hydrochloride (GdnHCl) on the phase transition temperature of poly (N-isopropylacrylamide) (PNIPAM) in aqueous solution with the aid of fluorescence spectroscopy, viscosity (η), and dynamic light scattering (DLS) measurements. Temperature dependent fluorescence intensity measurements have been employed successfully to determine the conformational change of PNIPAM through evaluating the Gibbs free energy changes. Additionally, direct hydrogen bonding formation between GdnHCl and PNIPAM has been confirmed by Fourier transform infrared (FTIR) spectroscopy measurements at various concentrations of GdnHCl. The intermolecular interactions were also studied in terms of amide I band analysis, which reveals the interruption of hydrated coil conformation of PNIPAM. The degree of destabilization of PNIPAM progressively increases with increasing concentration of the denaturant.