The interaction between Neutrophil Elastase (NE) and Toll-like receptor 4 (TLR4) has attracted substantial scientific attention, particularly regarding its potential role in cardiovascular diseases. Employing AlphaFold2, biomolecular docking, and MMGBSA calculation we aimed to predict their binding and validated the results through a co-immunoprecipitation study in a rat model with isoproterenol (ISO) -induced cardiac hypertrophy. Our findings strongly suggest a specific and plausible interaction between rat NE and rat TLR4, distinct from other neutrophil-derived serine proteases. Notably, AlphaFold2's precision was confirmed through cross-validation with known protein crystal structures, while Consurf analysis emphasized the evolutionary variable to conserve the rat NE - rat TLR4 binding site. HADDOCK, RosettaDock, ZDOCK, MD simulation, MMGBSA calculations, and superimposition with the stabilized structure complex all predicted strong binding between rat NE and rat TLR4. Our animal experiments revealed elevated NE and TLR4 expression in the hypertrophied myocardium following ISO infusion, with data confirming the physical interaction between NE and TLR4. Overall, this study sheds light on the intricate molecular association between NE and TLR4, underlining their potential significance in cardiovascular pathophysiology. Furthermore, it underscores AlphaFold2's reliability as a robust tool for predicting protein-protein interactions and complex structures.