Open-channel separation techniques, such as asymmetrical flow-field flow fractionation (AF4) and capillary electrophoresis (CE), can better preserve the fragile structures of biological complexes and accommodate much higher molecular weight analytes than conventional chromatography. Thus, my group has been applying AF4 and CE to analyze biological complexes. We have proved that AF4 can effectively separate the unbound and target-bound ssDNA during Systematic Evolution of Ligands by EXponential Enrichment (SELEX). This has led to the discovery of aptamers with high binding affinity and high efficacy in inhibiting target enzyme function through displacement of the natural ligand. We have also successfully coupled AF4 offline with CE to help obtain extracellular vesicles (EVs) free of contamination of matrix proteins, so that highly confident identification of the EV-specific protein cargos can be achieved. Both applications well demonstrate the power of open-channel separation techniques in rapid processing of biological complexes to permit downstream analysis of the involved molecular interactions.