Regenerative treatment for tympanic membrane perforation using Retympa® is a simple, minimally invasive treatment method that yields stable therapeutic outcomes, and is considered suitable for use in clinics. Herein, we report on cases treated at a clinic. The subjects were 38 patients and 40 ears treated at this clinic between July 2020 and November 2022. The patients ranged in age from 10 to 90 years old, with a mean age of 63.4 years, and included 14 male and 24 female patients. The most common etiology was chronic perforated otitis media, followed by post-placement of a tympanostomy tube. The perforations ranged in size from small to medium to large, in that order. The perforation closure rate with the first treatment with Retympa® was 67.5% (or 77.5% when cases that showed a marked reduction in size, but not complete closure, of the perforation were included). The closure rate with the first treatment was 71%, 78%, and 34% for small, medium, and large perforations, respectively. In this study, our investigation included cases that received treatment for both ears simultaneously, cases of post-operative perforation, cases with good hearing, and refractory cases. Retympa® is considered as an effective treatment modality that yields stable outcomes even in clinics.