Size-exclusion chromatography (SEC) associated with High Performance Liquid Chromatography (HPLC) is a powerful tool to separate, purify and fractionalize materials based on size. In this paper, we present a SEC method that was developed for the separation of thiol-capped Cd(Se,S) quantum dots (QDs) synthesized in the aqueous phase. A HPLC system with a SEC column and a cascade of three detectors (UV/Vis, FLD and ELSD) was used to analyze the different size fractions of QDs. Nanocrystals-HPLC column interactions were suppressed using thioglycolic acid (TGA) as an ion pair agent. Five fractions, namely F1 to F5, of different sizes and tunable optical properties were isolated from the original QDs sample. The emission peaks for fractions F1 and F2 were red-shifted and the fractions F4 and F5 were blue-shifted compared with the original sample, which suggested the presence of nanocrystals having different sizes. Dynamic Light Scattering (DLS) confirmed that collected fractions exhibited different hydrodynamic diameters ranging from 98.2 nm (fraction F1) to 24.9 nm (fraction F5). Also, fractions F2 and F4 were functionalized with glutathione and analyzed by HPLC-SEC. Glutathione-capped Cd(Se,S) QDs showed an increase in their molecular weight, when compared to bare TGA-capped Cd(Se,S) QDs, without a remarkable change of their crystal size and optical properties. The developed SEC technique allows a fast and reproducible separation of water-stable Cd(Se,S) QDs. Collected fractions with tunable optical properties could have potential applications in the nanotechnology area such as bio-imaging and diagnostics, e.g. cell sorting. © The Royal Society of Chemistry. 2012.