We have developed a quadrupole magnetic flow sorter (QMS) to facilitate high‐throughput binary cell separation. Optimized QMS operation requires the adjustment of three flow parameters based on the immunomagnetic characteristics of the target cell sample. To overcome the inefficiency of semiempirical operation/optimization of QMS flow parameters, a theoretical model of the QMS sorting process was developed. Application of this model requires measurement of the magnetophoretic mobility distribution of the cell sample by the cell tracking velocimetry (CTV) technique developed in our laboratory. In this work, the theoretical model was experimentally tested using breast carcinoma cells (HCC1954) overexpressing the HER‐2/neu gene, and peripheral blood leukocytes (PBLs). The magnetophoretic mobility distribution of immunomagnetically labeled HCC1954 cells was measured using the CTV technique, and then theoretical predictions of sorting recoveries were calculated. Mean magnetophoretic mobilities of (1−3) × 10−4 mm3/(T A s) were obtained depending on the labeling conditions. Labeled HCC1954 cells were mixed with unlabeled PBLs to form a “spiked” sample to be separated by the QMS. Fractional recoveries of cells for different flow parameters were examined and compared with theoretical predictions. Experimental results showed that the theoretical model accurately predicted fractional recoveries of HCC1954 cells. High‐throughput (3.29 × 105 cells/s) separations with high recovery (0.89) of HCC1954 cells were achieved.