Cells were separated with the aid of a multistep spiral fractionation device, utilizing hydrodynamic forces in a spiral tubing. The spiral was fabricated using "off-the-shelf microbore tubing, allowing for cheap and fast prototyping to achieve optimal cell separation. As a first step, a model system with 20 and 40 mu m beads was used to demonstrate the effectiveness of the multistep separation device. With an initial purity of 5%, a separation purity of 83% was achieved after a two-step separation with the addition of 0.1% polyethylene glycol (PEG)-8000. Next, doxorubicinresistant polyploid giant breast cancer cells (MDA-MB-231) were separated from doxorubicin-sensitive monoploid small breast cancer cells in the same fashion as the beads, resulting in a purity of around 40%, while maintaining a cell viability of more than 90%. Combined with basic cell analytical methods, the hydrodynamic separation principle of the device could be envisaged to be useful for a variety of cell fractionation needs in cell biology and in clinical applications.