Introduction: In reconstructive middle ear surgery, acoustic quality has received a high level of attention in recent years. Careful intraoperative selection and positioning of passive middle ear prostheses during tympanoplasty with ossiculoplasty is essential to ensure satisfactory sound transmission and postoperative hearing outcome. The reconstruction quality of the ossicular chain (OC) can be evaluated intraoperatively using a surgical assistance system which is based on a real-time monitoring system (RTM system) that acquires the transmission (middle ear transfer function (METF)) with electromagnetic excitation of the OC. In this experimental study, the METF with electromagnetic excitation of the (reconstructed) OC was compared to usual acoustic excitation and the benefit of the RTM system was investigated for the implantation of partial (PORP) and total (TORP) prostheses. Methods: Laser Doppler vibrometry (LDV) was used to measure the middle ear transfer function (METF) in 18 human temporal bones (TB). The RTM system uses electromagnetic excitation of the OC with a magnet placed on the umbo. Comparatively, measurements with the usual acoustical excitation, using an earphone in the external auditory canal, were performed. The measurements began with the intact OC, followed by real-time monitoring guided OC reconstruction with PORP and TORP. In addition, during the simulation of an intraoperative setting, the influence of the influence of opening (tympanomeatal flap lifted and pushed anteriorly) and closing (tympanomeatal flap folded back) the tympanic membrane on the measurements with the RTM system was determined. Results: Electromagnetic and acoustic excitation of the intact and the reconstructed OC provided comparable METF. The application of the RTM system significantly improved the quality of the OC reconstruction. The METF increased by up to 10 dB over the entire frequency range during implantation of the PORP with positioning control by the RTM system. When using the TORP, the METF could be improved by up to 15 dB. The opening of the tympanomeatal flap did not affect the measurements with the RTM system at the reconstructed OC. Conclusion: In this TB study, we demonstrated that the quality of OC reconstruction (improved METF as a benchmark for improved transmission) could be significantly increased by means of a RTM system. Intraoperative studies should now be conducted to investigate to which quantitative extent the intraoperative reconstruction quality can be improved and whether or not this improvement also manifests in an increased (long-term) hearing outcome. This will enable conclusions to be drawn about the contribution of the intraoperative reconstruction quality to the (long-term) hearing outcome in the context of the conglomerate of various factors influencing the postoperative hearing outcome.