The cochlea, a sensitive biosensor, detects sound waves and converts them into electrical signals the brain recognizes as sound. Damage to cochlear hair cells can release proteins, triggering biological responses that may impair hearing. Mass spectrometry-based proteomics offers insights into protein expression changes in cochlear tissues, improving our understanding of inner ear diseases. In this study, we performed a comprehensive proteomics analysis of whole cochlear tissue extracted from healthy guinea pigs and rats. The study optimized protein extraction protocols and analyzed cochlear protein expression using three biological replicates for each animal model. This work optimized protein extraction protocols and analyzed cochlear protein expression using three biological replicates for each animal model. The results identified 1801 proteins in guinea pigs and 3867 proteins in rats, with a high overlap in cochlear protein expression between the left and right ears—93% in guinea pigs and 89% in rats. The findings validate the assumption that the cochlear tissues from both sides of the ears can be considered biologically equivalent. This experiment provides a comprehensive cochlear proteome for guinea pigs and rats, supporting future studies on inner ear disorders.