Fractionation of α-lactalbumin (a-la) and β-lactoglobulin (β-lg), the 2 major whey proteins (WP), is quite challenging to scale-up by using eco-efficient technologies due to their similar molecular weight (14.2 and 18.36 kDa, respectively). The proposed approach was to evaluate the difference of baro-sensitivity of a-la and β-lg under high hydrostatic pressure (HHP) treatment and their capacity to form specific aggregates when combined to casein (CN), used as a ligand in this study. The objectives were to (1) evaluate the impact of pressure and time intensity on WP, (2) compare the use of 2 different type of CN: micellar (MC) and isoelectric (IC) as ligand to generate specific interactions with WP, and (3) determine optimal parameters of pressure/time combination and type of CN to improve WP fractionation. Model protein solutions composed of a-la, β-lg and CN (IC or MC) (2.5 mg/mL for each protein) were pressurized (200, 400 and 600 MPa during 100, 200 and 300 s) using an hydrostatic pressurization unit (Hiperbaric 135, Burgos, Spain). Proteins were characterized by HPLC and PAGE. Protein aggregation was studied (size and composition) using a high-performance size exclusion chromatography (HPSEC). Response surface model demonstrated that an optimal pressure/time of 600 MPa – 300s allowed to generate the highest a-la enriched fraction (85.04% and 79.79% purity with a protein recovery of 77.39% and 82.76%, respectively for IC and MC). Analysis by HPSEC showed that 2 main categories of aggregates were generated: (1) α_S1-CN, α_S2-CN and a large portion of β-lg mainly linked by disulfide bonds, and (2) α_S1-CN, α_S2-CN and a low amount of β-lg supposedly linked by hydrophobic interactions. Our experiments performed on dairy model solutions demonstrated that using HHP, an eco-efficient process, was suitable to generate specific aggregation of β-lg with CN ligand due to the difference of baro-sensitivity. Optimal pressurization parameters allowed to generate an a-la-enriched fraction with purity of 85.04%. The approach suggested that HHP could be used as pre-treatment for a new method of a-la fractionation from whey.