Ionic liquids (ILs) have found various applications in the field of biotechnology that involves protein extraction from the aqueous phase. However, the stability of biomolecules in ILs is still unpredictable. Therefore, this work aims to understand the effect of ammonium-based ILs with a fixed (trifluoromethylsulfonyl)imide [NTf2](-) anion and variable ammonium cations such as butyltrimethylammonium (IL-1), ethyldimethylpropylammonium (IL-2), diethylmethyl(2-methoxyethyl)ammonium (IL-3) and methyl-trioctylammonium (IL-4) on the stability of lysozyme. The spectroscopic analysis (UV, fluorescence and circular dichroism (CD)) revealed the existence of native structure of lysozyme in the presence of ILs at 25°C. Evidently, the presence of α-helix structure in lysozyme was confirmed using CD spectroscopy. In contrary, the thermal stability of the protein gradually decreased with increase in the concentration of the ILs. This was due to the strong favorable interactions of the ILs with the amino acid residues of the protein. Further, Nile red fluorescence revealed existence of the hydrophobic interactions between ILs and the lysozyme. Hence, due to its immense hydrophobic character, IL-4 thereby, decreased the catalytic activity and stability of the lysozyme to a greater extent.