Abstract  This study dealt with the partition behavior and partial purification of hexokinase (HK) from baker’s yeast by liquid-liquid extraction using aqueous two-phase polyethylene glycol (PEG)/citrate systems. First, we investigated the effect of agitation type (vortex and 8 rpm rotation) on the stability of the system, and then the effects of sodium citrate concentration, PEG concentration, and molar mass of PEG on the partition coefficient of this enzyme by using a 2⁵ factorial experimental design. The results of this factorial experiment showed the possibility of a partial purification of HK by using two extraction steps, since the enzyme preferentially migrated to the top phase and the total proteins (mainly contaminants) remained in the bottom phase. The purification factor (Pur _TOP) of the enzyme in the top phase was 1.87, and the partition coefficient of the total proteins (K _Prot ) was 0.47.