Despite its widespread prevalence, middle ear pathology, especially the development of proliferative lesions, remains largely unexplored and poorly understood. Diagnostic evaluation is still predicated upon a high index of clinical suspicion on otoscopic examination of gross morphologic features. We report the first technique that has the potential to non-invasively identify two key lesions, namely cholesteatoma and myringosclerosis, by providing real-time information of differentially expressed molecules. In addition to revealing signatures consistent with the known pathobiology of these lesions, our observations provide the first evidence of the presence of carbonate- and silicate-substitutions in the calcium phosphate plaques found in myringosclerosis. Collectively, these results demonstrate the potential of Raman spectroscopy to not only provide new understanding of the etiology of these conditions by defining objective molecular markers but also aid in margin assessment to improve surgical outcome.