BACKGROUND: The study aims to investigate the effect of stapedotomy on audiology measurements and the disease-specific health-related quality of life for patients with otosclerosis using the Danish Stapesplasty Outcome Test-25 as a quality of life measuring tool. METHODS: In this study, 50 patients who had undergone stapedotomy at our department between September 2017 and December 2020 were included. Data collection was performed by audiometric testing (pure tone and speech audiometry) in the pre-and postoperative settings. Health-related quality of life was assessed pre-and postoperatively in 30 patients using the validated Danish Stapesplasty Outcome Test-25. RESULTS: The mean improvement in air conduction thresholds was 27.7 dB and the mean improvement in air-bone gap was 21.8 dB. Health-related quality of life improved significantly after stapes surgery in all subscores of the Stapesplasty Outcome Test-25 (hearing, mental condition, social restriction, and general), except for “tinnitus.” The improvement in the audiometric data correlated significantly with the improvement in “total score” and “hearing function” but not with “tinnitus,” “social restrictions,” and “mental condition.” CONCLUSION: Stapedotomy leads to significant improvement in hearing and health-related quality of life. The Stapesplasty Outcome Test-25 can be used as a valuable supplement to the hearing test by assessing health-related quality of life.