This study was designed to show the changes in glycolmacropeptides (GMPs) of whey protein solution (WPS) due to different pretreatments before and after ultrafiltration (UF). The combined form of two variants (A&B) of GMPs is a helpful compound for nutritional management of phenylketonuria and ulcerative-colitis diseases and has low content of phenylalanine (Phe). WPS with 10% concentration was prepared, acidified (adjusted to pH = 3.0), and passed through a PES (polyethersulfone) membrane in the 1st-stage of ultrafiltration (UF-1). Then the resulting permeate was neutralized and went through the 2nd-stage of ultrafiltration (UF-2) under similar conditions. Four experiments of TRT-CON, CON-TRT, TRT-TRT, and CON-CON were used with different pretreatments, where TRT was a mixing-treatment of 30 min at 150 RPM applied either after acidification of WPS or after neutralization of first permeate and before UF-2 process. While the concentration and purity of the combined GMPs in UF-2 retentate in TRT-TRT respectively were >95.6 and 99.5%, its Phe became <10 ppm among the experiments. Highly glycolyzed polymers of GMPs (MW = 45–50 kDa) were formed in the TRT-TRT experiment and went through the pore sizes of PES membrane of UF-1 easily because of their flexible structure. However, they remained in the UF-2 retentate, due to to the formation of bulky polymers. The TRT-TRT experiment had the highest reversible and irreversible resistances for passing through the UF-1 and remaining on the UF-2 membranes, and its fouling index was significantly less than other experiments.