The practical problems and optimal conditions for partitioning of haemoglobin, and bovine serum albumin (BSA) from whole bovine blood using aqueous two-phase systems (ATPS) have been investigated. It was demonstrated that low molecular weight polyethylene glycol (PEG) seems to be better in terms of recovery and operability (such as low viscosity). The study revealed that only the sensitive area of the binodal has good potential in terms of total recovery of the proteins. Studies conducted with construction of ATPS with pre-dissolved and solid phase chemicals showed slight variation in the recovery of BSA and haemoglobin. In the case of ATPS constructed with solid phase chemicals, the top phase was pale red whereas it was pale yellow when using pre-dissolved phase chemical systems, possibly due to physical damage to the red blood cell (RBC) by the dissolution of the solid phase chemical present. The major proteins (free BSA, free haemoglobin) partition to the top and bottom phases, the cell debris and other contaminants partition at the inter-phase and the sediment is entirely RBC. (C) 2012 Elsevier B.V. All rights reserved.