Complex biological fluids, such as the vast and molecularly crowded cell cytoplasm and the highly viscoelastic mucus that protects many entry ways to the body, pose significant barriers to efficient gene delivery. Understanding the dynamics of gene carriers in such environments allows insight that leads to rational improvements in gene vector design. Fluorescence techniques that provide only ensemble-averaged transport characteristics do not provide detailed information related to the nature of various barriers to efficient gene vector transport to target cell nuclei. Multiple particle tracking (MPT) allows the tracking of the real-time motion of up to hundreds of individual particles simultaneously with high temporal and spatial resolution. We have adapted MPT to study gene carrier transport in live cells and in fresh, undiluted human mucus. By analyzing the displacements of gene vectors as a function of time scale, this technique provides, on a per particle basis, highly quantitative measurements of the transport rates and transport mechanisms, as well as biophysical information of the complex biological environments. Combining MPT with confocal microscopy (confocal particle tracking) allows dynamic and quantitative co-localization determination of gene carriers with various cellular structures, such as endosomes, lysosomes, the endoplasmic reticulum, and Golgi. We have applied MPT to enhance understanding of critical extracellular and intracellular bottlenecks to gene transfer.