The aim of this paper is to demonstrate the effects of hydrodynamic conditions on the permeate flux, cake resistance and sieving coefficient in a crossflow ultrafiltration process separating biomolecules of different molecular weights. A binary mixture of L-phenylalanine (L-phe) and lipase was ultrafiltered through a hydrophobic polyether sulphone (PES) membrane with 10 kDa molecular weight cut-off. The changes in permeate flux, cake layer resistance and observed sieving coefficient with different transmembrane pressures (TMP) and crossflow velocities were evaluated. The effect of TMP was examined at two different velocities (0.114 m s(-1) and 0.176 in s(-1)) and the effect of velocity was examined at two different TMP (20 kPa and 115 kPa) for the experimental system designed. In the initial stage of the crossflow filtration, it was determined that the TMP was more effective than the velocity. The cake layer resistance increased with increasing TMP and it decreased with increasing velocity for the high TMP value of 115 kPa. A maximum of the observed sieving coefficient was achieved with increasing velocity. An increase in TMP at low inlet velocity (0.114 in s(-1)) affected the observed sieving coefficient positively.