Intraoperative assessment of ossicular chain mobility remains challenging in reconstructive middle-ear surgery because conventional palpation is subjective. To better predict the postoperative air–bone gap (ABG) and guide decisions on whether revision surgery may be beneficial for hearing restoration, a more refined intraoperative assessment that differentiates the degree of ossicular chain mobility reduction is required. In this ex vivo study, we evaluated whether quasi-static stiffness measurements can predict sound transmission. Twelve human cadaveric temporal bones were mounted on a robotic setup in which a force-sensing needle robotically executed a standardized palpation sequence along a predefined trajectory, enabling calculation of direction-specific stiffness metrics. Stepwise artificial stiffening of the annular ligament, the stapes superstructure and the surrounding anatomical spaces of the tympanic cavity with thin layers of UV-curable resin produced graded sound transmission losses across up to five increments (for a total of ≈20–30 dB). The derived stiffness metrics tracked this decline only partially and anisotropically, with changes corresponding to <10 dB equivalents in specific probing directions. Nevertheless, binomial generalized linear models identified stiffness thresholds that discriminated among clinically relevant ABG categories (<10 dB, 10–20 dB, >20 dB). Similar trends were observed in three additional specimens with an opened incudostapedial joint. These finding demonstrate that quantitative force measurements or simplified surgical tools fitted to specific force thresholds could potentially provide surgeons with an intraoperative evaluation support tool.