A purification process of galacto-oligosaccharides (GOS), using three-stage serial nanofiltration (NF) units, operating under critical transmembrane pressure (TMPc) was designed. Three commercial flat-sheet membranes were tested at 53.5 °C and 65 °C, using GOS solutions at 20 and 40°Brix. Since one of the main drawbacks of NF for the purification of raw GOS is the removal of lactose, an enzymatic pre-hydrolysis step was also tested for producing a prehydrolyzed GOS and compared with non-hydrolyzed raw GOS. An empirical model showed that due to the increase in the solute concentration during batch NF, TMPc must be decreased with time to prevent fouling effects. Using NF serial system, the maximum GOS concentration achieved for raw GOS was 241 g/L and 156.8 g/L for pre-hydrolyzed GOS. Despite filtration times of the later were shorter, after three sequential batches, similar purities (∼55%) were obtained with both GOS solutions. Thus, the use of pre-hydrolyzed raw GOS as substrate for NF purification had two opposite effects: an improved flux due to lower lactose deposition on membrane, but also a lower GOS retention by the membrane, revealing a marginal gain in purification. The purities achieved were similar to those of commercial GOS, currently obtained by using conventional processes.