Engineered tendons and ligaments have great potential for the restoration of damaged tissues. Optimizing culture conditions to enhance their mechanical properties is critical for the development of these structural tissues. Ligaments’ cultivation within specialized bioreactors poses a challenge, especially due to the constraint surrounding the use of invasive methods, for mechanical characterization during growth. This paper introduces a non-invasive and non-destructive approach to quantify the mechanical properties of engineered ligaments in situ. The experimental approach involves acoustic excitation of the contained developing tissues using a speaker and employing Laser Doppler Vibrometry (LDV) to analyze resonant frequencies, mode shapes, and induced displacements. Preliminary experiments entail the use of synthetic strings suspended between movable clamps instrumented with force sensors. Acoustically induced tensions and motions of synthetic strings are measured using the force sensors attached to the string ends and LDV, respectively. The resonant frequency data for strings of various diameters at different preset tension levels match theoretical behavior well. The proof-of-concept experiments involve installing the synthetic strings into a tissue culture vessel and primarily using LDV to measure acoustically induced string dynamics to extract mechanical properties. Additional methods to validate our approach include digital holography (DH) and optical coherence tomography (OCT).