Multifocal microscopy (MUM), a technique to capture multiple fields of view (FOVs) from distinct axial planes simultaneously and on one camera, was used to perform micro-particle image velocimetry (µPIV) to reconstruct velocity and shear stress fields imposed by a liquid flowing around a cell. A diffraction based multifocal relay was used to capture images from three different planes with 630 nm axial spacing from which the axial positions of the flow-tracing particles were calculated using the image sharpness metric. It was shown that MUM can achieve an accuracy on the calculated velocity of around (0.52 ± 0.19) µm/s. Using fixed cells, MUM imaged the flow perturbations at sub-cellular level, which showed characteristics similar to those observed in the literature. Using live cells as an exemplar, MUM observed the effect of changing cell morphology on the local flow during perfusion. Compared to standard confocal laser scanning microscope, MUM offers a clear advantage in acquisition speed for µPIV (over 300 times faster). This is an important characteristic for rapidly evolving biological systems where there is the necessity to monitor in real time entire volumes to correlate the sample responses to the external forces.