Perfusion has been verified as one of efficient and economical choice for animal cell culture bioprocesses. Cell retention within the perfusion system determines the efficiency of the whole processes to a great extent. While the settling velocity of animal cells plays an important role in the design of the cell retention device and the estimation of settling operation, the measurement of this critical parameter suffers from the lack of simple and accurate method for a long time. In this work a novel method was developed from a simplified mathematical model, and then validated with a well-design settling experiment using standard micro-particles. Based on the measurement of the settling velocities of the hybridoma and hematopoietic cells, this method provides a series of valuable data that may be potential for the subsequent cell retention investigations.