Autoimmune sensorineural hearing loss (ASNHL) is the most common cause of sudden hearing loss in adults. Although the etiopathogenesis of this disease is unclear, it is widely believed that antibody and/or T-cell responses directed against inner ear-specific proteins may mediate ASNHL. Using the enzyme-linked immunospot (ELISPOT) assay, we have recently found that many patients with ASNHL have increased frequencies of peripheral blood T-cells capable of producing interferon (IFN)-γ in response to a homogenate of human inner ear tissue. Our studies may ultimately lead to the identification of inner ear-specific autoimmune targets in ASNHL, and our ELISPOT approach may be particularly useful in supporting the diagnosis of this disease entity. In the current chapter we detail how to use the ELISPOT assay for measuring frequencies of IFN-γ-producing T-cells in patients with ASNHL.