Choline chloride (ChCl) based deep eutectic solvents (DES) have become a designer solvent owing to their appealing properties and benign nature. The microscopic arrangement and properties of DES can be tailored by changing the molecular ratio of the constituent molecules. Low cost, easy handling, and low toxicity have enabled DES to emerge as a promising solvent media in the field of separation, synthesis, catalysis, and electrochemical process. Its remarkable physical properties and biocompatibility have caught the attention of various research groups leading to its wide-spread applications in diverse research fields, including the preservation of enzyme/protein activity. Most commonly used hydrogen bond donors like urea, ethylene glycol, malonic acid, etc. are taken in a specific ratio with ChCl to obtain these lower melting point mixtures. Several studies related to the morphological properties of ionic liquids (IL) guide the community regarding the wise use of the solvent, which is largely lacking in the case of DES. Reline is one of the most celebrated and highly used DESs, where urea (H2N-CO-NH2) is used as the hydrogen bond donor with choline chloride ([(CH3)3N-(CH2)2-OH] + Cl -) (ChCl) in 2:1 molar ratio. Apart from its biodegradability and economic viability, reline also has ample industrial applications. In particular reline has profound use in surface coating, biodiesel synthesis, and enzymatic reaction. Owing to its non-reactivity towards water, and acceptable toxicity limit, reline is used as a solvent for poorly water-soluble drugs. Recently, Monhemi and co-workers pointed out lower diffusivity of highly hydrogen-bonded urea in the proximity of Candida Antarctica lipase B that causes structural protection of the enzyme in the presence of ChCl forming a deep eutectic solvent. A similar kind of ultraslow solvent/cosolvent dynamics in the presence of reline is also established in our recent work, that effectively hinders the protein to deviate much from its equilibrium position. In this earlier work, we thoroughly analyzed the reline mediated structural protection of model protein HP-36. FTIR , time-resolved fluorescence spectroscopy have also been used to explore the stabilizing mechanism of reline. Choline cation has been combined with negatively charged amino acids to form a new class of IL, which has no environmental effects. Sahoo et al. in an experimental and computational approach, showed an enhanced stability of Cytochrome-c (Cyt-c) in the presence of amino acid-based biodegradable choline IL. Reline is also found to protect C-kit G-Quadruplex DNA from severely high temperature. Hydrogen bond mediated electrostatic interaction plays a pivotal role in stabilizing reline microstructure. ESPR, Neutron scattering, and molecular dynamics studies establish the position of the chloride ion at the closer proximity of urea and hydroxyl group of choline cation. The common outcome of all these studies is the hydrogen bond formation between the chloride ion and the urea molecule, which in turn accommodate the urea within the hydrogen bond network. Posada et al. confirmed an inhomogeneity in the structure of reline, despite the presence of the hydrophilic choline chloride and urea molecules. However, only few experimental and simulation studies demonstrated the role of the addition of water on the hydrogen-bonded network of reline. It has been shown that the viscosity, density, and refractive index of reline-water mixture are heavily disturbed In a recent work, Kashyap and group have reported a gradual modification of hydrogen bonds between the constituents of reline and water and found a drastic structural change of reline above the addition of 40wt% of water, which is in agreement with the neutron scattering study by Edler and coworkers. In another work, Kashyap and group have incorporated molecular dynamics simulation and reported the strengthening of the hydrogen bond between ethylene glycol and its subsequent nanosegregation on the addition of water and pointed out a competition between choline cation and ethylene glycol in making a hydrogen bond with the anion. Similar kind of competitive hydrogen bond formation in case of reline is also reported from the first principle molecular dynamics simulation by Siepmann and co-workers. The formation of three-dimensional hydrogen bonds predominantly makes the water structure tetrahedral. Conversely, the addition of osmolytes into water has an immense effect on the microscopic structure of water, for example: in a recent work, Smiatek and group proposed the strengthening of the water network in the presence of urea and TMAO. Besides that TMAO alone could also impact water network. Molecular dynamics simulations have shown that water-TMAO interaction is stronger than that of watertetramethylurea. This is in line with an earlier study by Paul et al. Despite having a similar structural framework, tert-butyl alcohol and TMAO show a drastic difference in their behaviors on the water structure. The presence of multiple potential hydrogen bonding sites is responsible for the interference of urea into the tetrahedral structure of water. In their investigation, Choudhury and group probed a range of aqueous solutions of urea from ~1M to ~9M and verified that even at very high concentration urea does not have its deleterious effect on the tetrahedrality of water. The same group in another work correlates the structural order and hydrogen bond network of water with varying temperature. This kind of behavior of urea in water is also reported by Ojha et al. in their recent work , though it is not in agreement with the Raman band analysis experiment by Walrafen et al., which predicts urea to be a structure breaker.