Complex biological environments, such as the cell cytoplasm or the mucus lining the airways of the lungs, can pose significant barriers to efficient therapeutic drug and gene delivery. Biological barriers are particularly important in controlled drug delivery applications that utilize a large carrier particle, such as a liposome or a polymer micro- or nanosphere. The dynamic transport of particulate drug and gene delivery vehicles through these barriers is poorly understood, having been primarily studied with static methods in the past. Recently, the transport of synthetic drug and gene carriers has been investigated quantitatively with real-time particle tracking technology, providing new insight into particle behavior in complex biological environments that is guiding rational improvements in particle design. This review briefly highlights basic principles of particle tracking and its application to elucidate important phenomena that limit effective particulate drug and gene delivery.