<p>This study presents an experimental methodology for estimating frequency-dependent damping in the human tympanic membrane (TM) using full-field time-domain holographic measurements and Short-Time Fourier Transform (STFT) analysis. Although damping plays a critical role in middle-ear mechanics, its experimental estimation remains challenging, with reported values exhibiting substantial variability. A high-speed digital holography (HDH) system is employed to capture transient displacement fields of the TM surface from cadaveric human temporal bones subjected to acoustic click excitation. The proposed approach enables the analysis of damping as a function of frequency by isolating free-vibration decay in the time–frequency domain, overcoming limitations of conventional time-domain techniques in systems with multiple overlapping modes. The results reveal a clear frequency dependence of effective system-level damping and its spatial variation across the membrane, as well as the influence of ossicular chain loading. This method offers a practical framework for extracting damping information from HDH experiments. The methodology provides robust local effective damping values relevant for improving the calibration and validation of finite element models of middle-ear mechanics.</p>