The downstream processing of enveloped virus‐like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl‐functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV‐1 gag virus‐like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 1012/mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double‐stranded DNA (88–90%) and host cell protein impurities (39–61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV‐1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 1011 particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 109 particles.