OBJECTIVES: Laser Doppler interferometry (LDI) of the eardrum allows noncontact optical analysis of its vibrations in response to sound. Although LDI has been widely used in research, it has not yet been introduced into clinical practice as an adjunctive test for otological workup. The aim of this study was to evaluate LDI as a diagnostic tool in the clinical sphere. STUDY DESIGN: Prospective. METHODS: A measurement system was developed based on a commercially available scanning He-Ne laser Doppler interferometer. The study included 129 eardrums of 79 subjects that were divided into 3 groups: 1) normal subjects and 2) patients with sensorineural and 3) conductive hearing loss (HL). All the patients suffering from conductive HL underwent ossiculoplasty, which allowed confirmation of the final diagnosis, and patients were assigned accordingly to the subgroups malleus fixation, incus luxation, and stapes fixation. RESULTS: The modified LDI system allowed bilateral evaluation of a subject within 30 minutes. No significant difference between normal subjects and patients having sensorineural HL were found. However, it was possible to distinguish between normal subjects and patients with conductive HL. Furthermore, the system had the ability to differentiate between various middle ear diseases. These groups differed statistically significantly in terms of manubrium vibration amplitude and resonance frequency. In malleus fixation significant differences in tympanic membrane movement patterns were found. CONCLUSIONS: Our LDI is applicable in clinical otological practice and serves as a valuable addition to the routine audiological investigations for preoperative evaluation of the mobility and integrity of the ossicular chain.