The etiology of an asymmetric sensorineural hearing loss can often be difficult to determine. Because a wide variety of pathologic processes may be responsible for the hearing loss, numerous diagnostic tests are usually used in the initial evaluation, including pure-tone audiometry, acoustic reflex testing, imaging, serologic testing, and auditory brainstem response testing. The diagnostic evaluations of 225 consecutive cases of asymmetric sensorineural hearing loss are reviewed. A cochlear site-of-lesion was demonstrated in the majority (194) of patients. Because all retrocochlear lesions (31) were associated with an abnormal auditory brainstem response, imaging should be performed in that group of patients. Magnetic resonance imaging offers greater specificity than computed tomography. Reflex decay, acoustic reflex testing, and rollover were all associated with a high false-negative rate. Whereas serologic testing for syphilis yielded several cases of otosyphilis, thyroid function testing was of little value. A diagnostic protocol for asymmetric sensorineural hearing loss is presented.