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Single particle profiling (SPP) is a unique methodology to study nanoscale bioparticles such as liposomes, lipid nanoparticles, extracellular vesicles, and lipoproteins in a single particle and high throughput manner. The initial version requires the single photon counting modules for data acquisition, which limits its adoptability. Here, we present imaging-based SPP (iSPP) that can be performed by imaging a spot over time in the common imaging mode with confocal detectors. We also provide user-friendly software with a graphical user interface to analyze such data and give quantitative insights on the content and properties of nanoscale bioparticles. We use iSPP to decipher lipid-protein interactions, membrane modifications by drugs, and the heterogeneity of extracellular vesicles isolated from cell lines and human urine. This easily applicable modality of the single particle profiler will facilitate nanoscale bioparticle research in laboratories with access to any confocal microscope.