Significance: Worldwide, ∼460  million people suffer from disabling hearing impairment. Many of these patients are still not sufficiently supplied with currently available auditory technologies. Optical stimulation of the hearing organ offers a promising alternative for a new generation of auditory prostheses.

Aim: To assess the biocompatibility margins of our laser pulse amplitude strategy in vitro, we designed a protocol and present the effects on normal human dermal fibroblasts, human chondrocytes, and human osteoblasts.

Approach: Laser pulses of 532 nm were applied over 120 s using our laser pulse amplitude modulation strategy. We then assessed cell viability and cytotoxicity through fluorescence staining and quantitative polymerase chain reaction-analysis regarding 84 key player-genes for cytotoxicity and stress response.

Results: The first in vitro biocompatibility margins for our stimulation parameters applied to cells of the peripheral hearing organ were between 200 and 223 mW (3348  J/cm²). After irradiation with a subphototoxic laser power of 199 mW (2988  J/cm²), only the fibroblasts showed a significant upregulation of GADD45G.

Conclusion: Further studies are underway to optimize parameters for the optoacoustic stimulation of the auditory system. Our protocol and results on laser–tissue interactions can be useful for translational laser applications in various other irradiated biological tissues.