Eukaryotic cells contain actin filaments and microtubules that function as cytoplasmic filaments for vesicle transport. The study of vesicle transport on these filaments in vitro advanced rapidly when vesicle transport assays were developed. High-resolution multimode video-enhanced microscopy is the method used for such assays to detect vesicles as small as 100 nm in diameter and microtubules 25 nm in diameter. Vesicle transport is recorded in real time on high-capacity digital storage media. Motion analysis is performed to quantify different parameters of vesicle movement along cytoplasmic filaments including velocity, distances travelled and straightness of path. In vitro vesicle transport assays proved highly successful and enabled the identification of important factors that are essential for regulation of intracellular transport.