The transport of magnetic particles (MPs) by dynamic magnetic field landscapes (MFLs) using magnetically patterned substrates is promising for the development of Lab-on-a-chip (LOC) systems. The inherent close-to-substrate MP motion is sensitive to changing particle–substrate interactions. Thus, the detection of a modified particle–substrate separation distance caused by surface binding of an analyte is expected to be a promising probe in analytics and diagnostics. Here, we present an essential prerequisite for such an application, namely the label-free quantitative experimental determination of the three-dimensional trajectories of superparamagnetic particles (SPPs) transported by a dynamically changing MFL. The evaluation of defocused SPP images from optical bright-field microscopy revealed a “hopping”-like motion of the magnetic particles, previously predicted by theory, additionally allowing a quantification of maximum jump heights. As our findings pave the way towards precise determination of particle–substrate separations, they bear deep implications for future LOC detection schemes using only optical microscopy.