Ions play a key role in the destabilization of collagen. This study explores the effect of diethyl methyl ammonium methane sulfonate (AMS), an ionic liquid (IL), on different hierarchical orderings of collagen, namely, at the molecular and fibrillar levels. The rheological behavior and secondary structural changes reveal changes in the hydrogen bonding environment of collagen, leading to alterations in the triple helical structure of collagen. An increase in the concentration of AMS resulted in swelling of rat-tail tendon fibers, and also, decreased thermal stability signifies that ions are obliged to destabilize collagen at the fibrillar level. Molecular modeling studies confirm that anions are judiciously held responsible for structural deformities in collagen, whereas cations have a tenuous effect. Thus, the preferential role of ions present in an ammonium IL has been elucidated in this study.