Bioactive oligosaccharides obtained either from natural or synthetic sources are normally present in highly complex matrices.This heterogeneity is particularly important at the carbohydrate fraction.In this context, the suitable fractionation of bioactive oligosaccharides becomes an important task to broaden their applications, as well as to accomplish their structural and/or functional characterization.This chapter deals with a wide number of techniques at semi-and preparative scale, i.e. membrane separation processes, chromatographic techniques, solvent-based methods, microbiological and enzymatic techniques, whose applications largely depend on the use and the purity required for the fractionated oligosaccharides.The advantages and major drawbacks of the different techniques are also discussed in detail.In addition to well-established techniques, such as ultrafiltration, nanofiltration, size-exclusion or ion exchange chromatography, this chapter will also describe novel and green techniques which have been recently reported to be useful for the prospective fractionation of oligosaccharides with high recoveries, including pressurized liquid extraction, supercritical fluid and ionic liquids.