Lysosomal storage diseases (LSDs) are genetic disorders caused by enzyme deficiencies that lead to lysosomal dysfunction and progressive cell damage. Accurate visualization and quantification of lysosomal morphology and subcellular localization are essential parameters for understanding the pathology and disease progression of different LSDs, as well as for developing effective therapies. Here, we successfully identified and characterized lysosomes using a holo-tomographic flow cytometry (HTFC) technique, which allows for label-free, high-content, and high-throughput 3D imaging of lysosomal compartments in single live cells. This study could complement traditional gold-standard methods to overcome the actual limitations. Leveraging this technology, we propose quantitative biomarkers of lysosomal accumulation in LSD-affected cells. In fact, by generating refractive index tomograms, we achieved accurate measurement and comprehensive 3D visualization of cytoplasmic lysosomal aggregation in suspended single cells. Through experimental validation and advanced computational analyses, we identified a quantitative correlation between the 3D lysosomal architecture and the efficacy of various therapeutic strategies, including genetic and pharmacological interventions. This work represents a significant advance in lysosomal research and may support future efforts to improve diagnostics and develop targeted therapies for LSDs.