The urea molecule is a well-known denaturant for a wide range of macromolecules. To date, there is no unified molecular-level explanation for urea-induced denaturation of all macromolecules. As a result, considerable effort has been directed toward this subject in recent years, because osmolyte protein interactions are of central interest and have implications ranging from polymer physics to cell biology. Detailed urea denaturation mechanisms, focusing on the entropically driven formation of urea clouds, urea induced cross-linking mechanism, and osmolyte clouding around macromolecules, have been proposed in the literature. However, no agreement has been reached on the molecular machinery of urea denaturation. In this thesis, the urea-induced solubility changes of macromolecules in aqueous solutions were investigated by utilizing the lower critical solution temperature (LCST) of poly (N - isopropylacrylamide) (PNIPAM) and poly (N,N-diethylacrylamide) (PDEA) as a function of urea concentration up to 6.0 M. Due to the lack of suitable probing methods for the collapsed state of interested macromolecules, a temperature-controlled ATR – FTIR spectroscopy based method was developed to explore the interaction between urea and the collapsed state of macromolecule. LCST measurements revealed that the solubility of PDEA increases with increasing urea concentrations, whereas PNIPAM salts-out from the solution monotonically, despite the fact that both polymers have similar molecular structures. Temperature gradient ATR-FTIR measurements were carried out to further investigate this discrepancy. First, no favourable urea accumulation towards the collapsed form of macromolecules was observed up to 6.0 M urea, which puts doubt on the urea clouding mechanism. Moreover, at elevated (> 3.0 M) urea concentrations, the collapsed form of the PNIPAM and PDEA accumulated towards the cooler parts of temperature gradient in solution, indicating the preferred form of the macromolecule is the soluble (uncollapsed) form. These surprising results indicate that both PNIPAM and PDEA prefers the soluble form at elevated urea concentrations above the LCST. As a molecular mechanism, the urea molecules act as a "glue" between the amide groups of PNIPAM, bringing intra- and inter-molecular parts of the macromolecule into close proximity, and act as a collapsed form of macromolecules. Apparently, such a mechanism is not valid for the PDEA. Our findings shed new light not only on aqueous phase phenomena, but also on the aggregated (collapsed) phase of macromolecules in aqueous medium.