Accurate diagnosis of most middle ear pathological conditions including otitis media remains challenging in a clinical setting. Standard ear evaluations are still performed primarily on the basis of human recognition of morphologic patterns in vivo using white light otoscope and suffer from significant observer variability providing minimal understanding of a disease’s underlying biochemistry. Owing to the biochemical specificity and multiplexing capability, spectroscopy-based chemical imaging has emerged as a promising tool for middle ear disease diagnosis in a real-time, label-free and non-destructive manner. Here, we critically review the spectroscopy-based approaches employed to study middle ear pathological conditions.