Beta-2 transferrin is a protein found in cerebral spinal fluid and inner ear perilymph, but not in blood, nasal, or ear secretions. The clinical use of this test has been previously demonstrated, but sources of test error have not been addressed. The purpose of this study was to evaluate sources of error related to this test in order to improve its clinical use. We reviewed the specimens submitted for beta-2 analysis over the first 12 months of test availability at our institution to identify potential factors leading to test error. Sources of error were categorized into the following groups: sample collection, delivery, and extraction factors; assay factors; physician-related factors; and patient-related factors. The test for beta-2 transferrin is a valuable diagnostic tool for the management of difficult clinical problems, provided the physician is aware of potential factors that can lead to test error and clinical mismanagement.