Introduction: Scientific and clinical interest in extracellular vesicles (EVs) has increased rapidly as evidence mounts that they may constitute a new signaling paradigm. Recently, it is well recognised that cells secrete essentially two EV subtypes that can be partially separated by differential centrifugation: the larger size class (sMVs) are heterogeneous, while the smaller size class (exosomes) are relatively homogeneous in size. A key issue hindering progress in understanding underlying mechanisms of EV biogenesis and cargo selectivity has been the technical challenge of isolating homogeneous EV sub-populations suitable for molecular analysis. Methods: In this study we reveal a novel method, based on sequential centrifugal ultrafiltration (SCUF), affords unbiased isolation of EVs from cell medium from the human colon cancer cell LIM1863 as a model system Characterize both EV subtypes using dynamic-light scattering, cryo-electron microscopy, immunoblotting, comparative label-free quantitative proteome profiling (spectral counting, Mascot, Scaffold), in addition to fibroblast invasion. Result: Comparative protein profiling of SCUF-Exos and sMVs reveals 354 and 608 unambiguous protein identifications, respectively, with 256 proteins in common. A salient finding was the first report of 350 proteins uniquely identified in sMVs may of which have the potential to enable discrimination of this EV types from exosomes. We also reveal TSG101, Alix/PDCD6IP, and CD63, considered stereotypic protein markers of exosomes, to be exclusively enriched only in exosomes, while numerous proteins found exclusively in sMVs (KIF23, RACGAP1, CSE1L/CAS). We also reveal numerous proteins found exclusively in sMVs. Summary/conclusion: This analytical SCUF method developed is potentially scalable using tangential flow filtration and provides a solid foundation for future in-depth functional studies of EV subtypes from diverse cell types.