A new cell analysis method, differential mobility cytometry (DMC), was developed to monitor cells spatially and temporally or to separate cells based on affinity interactions. DMC combines an oscillation system with open-tubular capillary cell affinity chromatography (OT-CAC), although any separation volume (capillaries, channels, etc.) can be used. This unique separation approach uses oscillating flow and differential imaging to analyze cells as they retard and adhere to an affinity surface. Three main factors of the oscillation system were studied: the pump speed, oscillation frequency, and cell velocity at different oscillation speeds. The optimized oscillation frequency and intensity were determined. Cell-surface interactions were used to estimate the number of bonds formed during cell capture. An average of 200 bonds (standard deviation of 150 bonds) were formed during cell capture. The variability was due to differences in cell-capture times (0.8 ± 0.6 s). Cells expressing the target protein on the surface oscillated slower and were captured by the corresponding ligand on the capillary surface. Cells were detected by differential imaging of a charge-coupled device camera. DMC measurements were optimized with respect to the camera frame difference. Cells were observed to slow as they reached the surface and could be observed to sway in the oscillating flow as they were tethered to the surface by a capture antibody. With the advantage of high cell-capture efficiency and temporally monitoring cell adhesion by the differential mobility of cells, DMC has proven to be a useful tool in cell analysis for basic biological studies and biomedical research.