<p>Reliable assessment of 3D stem cell spheroid function and fate is crucial in regenerative medicine and cell therapy, where gene and protein analyses and mechanical probing are employed. However, current methods are often invasive and struggle with heterogeneity, frequently damaging spheroid integrity, which drives a need for non-destructive, label-free techniques to assess individual spheroids directly in their culture environment. Here, we present a label-free platform, UNIQUE (Ultrasound Non-destructive In-situ Quantitative Evaluation), for single-spheroid, in-situ quantification of stem cell spheroid deformability as a mechanical phenotype during differentiation into specific lineages. We established UNIQUE using focused ultrasound to non-invasively assess stem cell spheroid deformability under optimized conditions (3 MPa, 30% duty cycle, surface alignment). We measured changes in spheroid deformability in real time as they differentiated into specific lineages. We also achieved label-free classification of adipogenic spheroids using acoustic property-based spheroid trapping. Our UNIQUE solution provides a transformative framework for dynamic metabolic profiling and better-quality control in the manufacturing of stem cell spheroids.</p><p></p>